Branched cyclic tetrasaccharide, process for producing the same, and use

ABSTRACT

The object of the present invention is to provide a novel glycosyl derivative of cyclotetrasaccharide represented by cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}, and it is solved by providing a branched cyclotetrasaccharide, wherein one or more hydrogen atoms in the hydroxyl groups of cyclotetrasaccharide are replaced with an optionally substituted glycosyl group, with the proviso that, when only one hydrogen atom in the C-6 hydroxyl group among the above hydrogen atoms is substituted with an optionally-substituted glycosyl group, the substituted glycosyl group is one selected from those excluding D-glucosyl group.

TECHNICAL FIELD

[0001] The present invention relates to a novel branched cyclictetrasaccharide, more particularly, a glycosyl derivative of a cyclictetrasaccharide represented by the chemical formula ofcyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→},a process for producing the same, and uses thereof.

BACKGROUND ART

[0002] α-, β, and γ-Cyclodextrins, which consist of 6, 7, and 8 glucosemolecules that are linked each other via the α-1,4 glucosyl linkage,respectively, have been known as cyclic saccharides composed of glucoseunits. These cyclodextrins have been used in a variety of fields due totheir advantageous inherent properties of non-reducibility, tasteless,enclosing hydrophobic materials, etc. There have been being pursuedremarkable researches directed to improve properties of cyclodextrinsand impart additional new functions thereupon. For example, JapanesePatent Kokai Nos. 9,708/94, 14,789/94, 16,705/94, 298,806/94, and25,305/98 proposed branched cyclodextrins with different branchingstructures, which are prepared by coupling a glycosyl group such as aglucosyl, galactosyl, mannosyl, glucosaminyl, or N-acetylglucosaminylgroup to cyclodextrins; processes for producing the same; and usesthereof.

[0003] As an example of cyclic saccharide reported recently, there is acyclic tetrasaccharide, reported by Gregory L. Cote et al. in EuropeanJournal of Biochemistry, Vol. 226, pp. 641-648 (1994), composed ofglucose molecules linked each other via the alternating α-1,3 and α-1,6bonds and having the structures represented by Chemical Formulae A and Bas bonding fashions between atoms and between glucosyl groups,respectively. In addition to Chemical Formulae A and B,cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1-3)-α-D-glucopyranosyl-(1→}belongs to the above cyclic tetrasaccharide. Throughout thespecification, the term “cyclotetrasaccharide” means theabove-identified cyclic tetrasaccharide.

[0004] Chemical Formula B:

cyclo{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→}

[0005] The above report by Cote et al. shows that cyclotetrasaccharideis formed by allowing alternan, a type of polysaccharide composed ofglucose molecules linked via the alternating α-1,3 and α-1,6 bonds toact on alternanase, a type of hydrolyzing enzyme. Thereafter,cyclotetrasaccharide has been expected to be used in different fieldssimilarly as in or much more useful than conventional cyclodextrins. Themethod of the report, however, may not suitable for an industrial-scaleproduction of cyclotetrasaccharide, because alternan used as a startingmaterial is not easily obtainable and the yield of cyclotetrasaccharidefrom the material is insufficient in view of industrial-scaleproduction.

[0006] The same applicant as the present invention disclosed anα-isomaltosyl-transferring enzyme, a novel enzyme which formscyclotetrasaccharide when acts on a saccharide having a glucosepolymerization degree of at least three and having both an isomaltosylgroup at the non-reducing end and the α-1,4 glucosyl bond as a linkageother than the linkage at the non-reducing end (abbreviated as“α-isomaltosylglucosaccharide” hereinafter) as disclosed in JapanesePatent Application No. 149,484/2000, and Japanese Patent Application No229,557/2000 (International Publication No. WO 01/90,338 A1) applied forbased on the above Japanese Patent Application; and also disclosed anα-isomaltosylglucosaccharide-forming enzyme, a novel enzyme which formsα-isomaltosylglucosaccharide when acts on a maltooligosaccharide havinga glucose polymerization degree of at least three, as disclosed inJapanese Patent Application No. 233,364/2000 and Japanese PatentApplication No. 234,937/2000 (International Publication No. WO 02/10,361A1), applied for based on Japanese Patent Application No.233,364/2000.Further, the above applicant proposed a method to producecyclotetrasaccharide as a main product from starch, a widely, commonlyused material for producing foods by using theα-isomaltosylglucosaccharide-forming enzyme and theα-isomaltosyl-transferring enzyme in combination, as disclosed inJapanese Patent Application Nos. 233,364/2000 and 234,937/2000(International Publication No. WO 02/10,361 A1). This proposal was abreakthrough for an industrial scale production of cyclotetrasaccharide.

[0007] Thus, the study on cyclotetrasaccharide has just merely beenstarted, and further studies for elucidating unknown functions anddeveloping new uses of cyclotetrasaccharide are now being greatlyexpected. Even though cyclotetrasaccharide is a known compound, there isfound no study to produce derivatives thereof as a main object, becausecyclotetrasaccharide has not yet been easily obtained. So far found ismerely the above report by Cote et al. that reported only a6-O-glucopyranosyl derivative of cyclotetrasaccharide, represented byChemical Formula C, isolated and identified as a by-product in anegligible yield through the action of alternanase on alternan. ChemicalFormula D represents the 6-O-glucopyranosyl derivative in terms ofbonding fashions between glucosyl groups.

[0008] Similarly as in cyclodextrins, supplying of glycosyl derivativesof cyclotetrasaccharide would provide a useful knowledge for developinguses of cyclotetrasaccharide through analyses on their properties, andalso it remarkably influences on the development of uses of novelsaccharides, obtained by improving or modifying the properties andfunctions of cyclotetrasaccharide.

DISCLOSURE OF INVENTION

[0009] In view of the above backgrounds, the first object of the presentinvention is to provide a novel glycosyl derivative ofcyclotetrasaccharide, the second object is to provide a process forproducing the same, and the third object is to provide uses thereof.

[0010] To solve the above objects, the present inventors firstly foundthat cyclotetrasaccharide-related-saccharides were formed as by-productsin the reaction system accomplished by the present inventors, wherecyclotetrasaccharide was formed by subjecting a partial starchhydrolyzate to the action of α-isomaltosyl-transferring enzyme andα-isomaltosylglucosaccharide-forming enzyme, and then tried to isolateand identify the by-products. As a result, they confirmed that all theby-products were novel glycosyl derivatives of cyclotetrasaccharide.Further, they reacted cyclotetrasaccharide, prepared by the abovetwo-types of enzymes, with these enzymes together with well knownsaccharide-relatedenzymes in the presence of different glycosyl donors.As a result, they found that various glycosyl derivatives were obtainedthrough the action of the above enzymes, cyclomaltodextringlucanotransferase, β-galactosidase, α-galactosidase, lysozyme, andother saccharide-related-enzymes such as glycosyltransferase,glycosylhydrolase, and glycosylphosphatase. They isolated the formedglycosyl derivatives of cyclotetrasaccharide and examined theirproperties, and confirmed that the glycosyl derivatives can beadvantageously used in the fields of food products, cosmetics,pharmaceuticals, etc. The present invention was made based on the aboveself-findings by the present inventors.

[0011] The present invention solves the first object by providingbranched cyclotetrasaccharides, i.e., glycosyl derivatives ofcyclotetrasaccharide represented by Formula 1.

[0012] wherein in Formula 1, R₁ to R₁₂ each independently represents anoptionally substituted glycosyl group or hydrogen atom, with the provisothat all of R₁ to R₁₂ are not hydrogen atom at the same time and that,when either R₄ or R₁₀ is an optionally substituted glycosyl group, theglycosyl group R₄ or R₁₀ is a glycosyl group other than D-glucopyranosylgroup.

[0013] The present invention solves the second object by providing aprocess for producing the branched cyclotetrasaccharides of the presentinvention, which uses an enzyme capable of transferring a glycosyl groupfrom a monosaccharide, oligosaccharide, or polysaccharide tocyclotetrasaccharide; and comprises a step of forming the branchedcyclotetrasaccharides by reacting the above enzyme with a mixture ofcyclotetrasaccharide and the above monosaccharide, oligosaccharide, orpolysaccharide, and collecting the formed branchedcyclotetrasaccharides.

[0014] Further, the present invention solves the third object byproviding a composition in the form of a food product, cosmetic, orpharmaceutical, which comprises the branched cyclotetrasaccharide(s) ofthe present invention.

BRIEF DESCRIPTION OF DRAWINGS

[0015]FIG. 1 is a chromatogram of cyclotetrasaccharide onhigh-performance liquid chromatography (abbreviated as “HPLC”hereinafter).

[0016]FIG. 2 is a ¹H-NMR spectrum of cyclotetrasaccharide.

[0017]FIG. 3 is a ¹³C-NMR spectrum of cyclotetrasaccharide.

[0018]FIG. 4 is a ¹³C-NMR spectrum of the branched cyclotetrasaccharideof the present invention, represented by Chemical Formula 1.

[0019]FIG. 5 is a ¹³C-NMR spectrum of the branched cyclotetrasaccharideof the present invention, represented by Chemical Formula 3.

[0020]FIG. 6 is a ¹³C-NMR spectrum of the branched cyclotetrasaccharideof the present invention, represented by Chemical Formula 4.

[0021]FIG. 7 is a ¹³C-NMR spectrum of the branched cyclotetrasaccharideof the present invention, represented by Chemical Formula 5.

[0022]FIGS. 8a and 8 b are respectively a chromatogram (a) on HPLC for areaction mixture obtained by reacting CGTase with a mixture ofcyclotetrasaccharide and α-cyclodextrin, and a chromatogram (b) on HPLCfor a reaction mixture obtained by contacting glucoamylase with theabove mixture after reacted with CGTase.

[0023]FIG. 9 is a ¹³C-NMR spectrum of the branched cyclotetrasaccharideof the present invention, represented by Chemical Formula 2.

[0024]FIG. 10 is a ¹³C-NMR spectrum of the branched cyclotetrasaccharideof the present invention, represented by Chemical Formula 6.

[0025]FIG. 11 is a ¹³C-NMR spectrum of the branched cyclotetrasaccharideof the present invention, represented by Chemical Formula 8.

[0026]FIG. 12 is a ¹³C-NMR spectrum of the branched cyclotetrasaccharideof the present invention, represented by Chemical Formula 7.

[0027]FIG. 13 is a ¹³C-NMR spectrum of the branched cyclotetrasaccharideof the present invention, represented by Chemical Formula 9.

[0028]FIG. 14 is a ¹³C-NMR spectrum of the branched cyclotetrasaccharideof the present invention, represented by Chemical Formula 10.

[0029]FIG. 15 is an X-ray diffraction spectrum for a crystal of thebranched cyclotetrasaccharide of the present invention, represented byChemical Formula 1.

[0030]FIG. 16 is an X-ray diffraction spectrum for a crystal of thebranched cyclotetrasaccharide of the present invention, represented byChemical Formula 2.

[0031]FIG. 17 is an X-ray diffraction spectrum for a crystal of thebranched cyclotetrasaccharide of the present invention, represented byChemical Formula 3.

[0032]FIG. 18 is an X-ray diffraction spectrum for a crystal of thebranched cyclotetrasaccharide of the present invention, represented byChemical Formula 6.

[0033]FIG. 19 is an X-ray diffraction spectrum for a crystal of thebranched cyclotetrasaccharide of the present invention, represented byChemical Formula 7.

[0034]FIG. 20 shows a thermal property of the branchedcyclotetrasaccharide of the present invention, represented by ChemicalFormula 1 on thermogravimetric analysis.

[0035]FIG. 21 shows a thermal property of the branchedcyclotetrasaccharide of the present invention, represented by ChemicalFormula 2 on thermogravimetric analysis.

[0036]FIG. 22 shows a thermal property of the branchedcyclotetrasaccharide of the present invention, represented by ChemicalFormula 3 on thermogravimetric analysis.

[0037]FIG. 23 shows a thermal property of the branchedcyclotetrasaccharide of the present invention, represented by ChemicalFormula 6 on thermogravimetric analysis.

[0038]FIG. 24 shows a thermal property of the branchedcyclotetrasaccharide of the present invention, represented by ChemicalFormula 7 on thermogravimetric analysis.

BEST MODE FOR CARRYING OUT THE INVENTION

[0039] Preferred embodiments of the present invention are describedbelow in more detail:

[0040] 1. Branched Cyclotetrasaccharides

[0041] Novel branched cyclotetrasaccharides, which the present inventionprovides, have the structure represented by Formula 1.

[0042] wherein in Formula 1, R₁ to R₁₂ each independently represents anoptionally substituted glycosyl group or hydrogen atom, with the provisothat all of R₁ to R₁₂ are not hydrogen atom at the same time and thatwhen either R₄ or R₁₀ is an optionally substituted glycosyl group, theglycosyl group R₄ or R₁₀ is a glycosyl group other than D-glucopyranosylgroup. The term “glycosyl group” as referred to as in the presentinvention means an atomic group represented by a structure where ananomeric hydroxyl group is removed from the molecular structure of asaccharide. The term “saccharides” as referred to as in the presentinvention means a general term for compounds including polyalcohols andtheir aldehydes, ketons and acids; amino sugars and their derivatives;and oligosaccharides, polysaccharides, and their condensed compounds.The term “substituents in glycosyl groups with an optional group” asreferred to as in the present invention means substituent groups whichcan substitute hydrogen(s) of one or more non-anomeric hydroxyl groupsin a saccharide molecule, or of one or more non-anomeric hydroxyl groupsand amino groups when the saccharide molecule is an amino sugar.Examples of such substituent groups are alkyl, acyl, acetyl, phosphoricacid, and sulfuric acid groups.

[0043] Examples of the glycosyl groups that are positioned at thebranched parts of the branched cyclotetrasaccharides of the presentinvention, i.e., one or more glycosyl groups selected from R₁ to R₁₂ inFormula 1 are optionally substituted {α-D-glucopyranosyl-(1→4)-}_(n)α-D-glucopyranosyl groups, with the proviso that “n” represents aninteger of 0 or over and, when at least two of R₁ to R₁₂ are the aboveglycosyl groups, the integers of “n” in each glycosyl groups areindependent each other; optionally substitutedα-D-glucopyranosyl-(1→6)-{α-D-glucopyranosyl-(1-3)-α-D-glucopyranosyl-(1→6)-}_(n)α-D-glucopyranosyl group, with the proviso that “n” represents aninteger of 0 or over and, when at least two of R₁ to R₁₂ are the aboveglycosyl groups, the integers of “n” in each glycosyl groups areindependent each other; optionally substituted{β-D-galactopyranosyl-(1→6)-}_(n)β-D-galactopyranosyl groups, with theproviso that “n” represents an integer of 0 or over and, when at leasttwo of R₁ to R₁₂ are glycosyl groups, the integers of “n” in eachglycosyl groups are independent each other; optionally substitutedα-D-galacropyranosyl groups; and optionally substituted β-D-chitosaminylgroups. The branched cyclotetrasaccharides of the present invention mayhave one or more of the above-mentioned groups intramolecularly.

[0044] The first more concrete example of the branchedcyclotetrasaccharides are those wherein R₁ and/or R₇ in Formula 1 areoptionally substituted {α-D-glucopyranosyl-(1-4)-}_(n)α-D-glucopyranosyl groups, with the proviso that “n” represents aninteger of 0 or over and, when both R₁ and R₇ are such glycosyl groups,the integers of “n” in each groups are independent each other. Examplesof the structural formulae thereof are Chemical Formulae 1 and 2 asshown in Experiments 3-4 and 4-3.

[0045] The second more concrete example of the branchedcyclotetrasaccharides are those wherein R₂ and/or R₈ in Formula 1 areoptionally substitutedα-D-glucopyranosyl-(1→6)-{α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-}aα-D-glucopyranosyl groups, with the proviso that ‘n’ represents aninteger of 0 or over and, when both R₂ and R₈ are such glycosyl groups,the integers of “n” in each groups are independent each other. Examplesof the structural formulae thereof are Chemical Formulae 3 and 4 asshown in Experiment 3-4.

[0046] The third more concrete example of the branchedcyclotetrasaccharides are those wherein R₂ and/or R₈ in Formula 1 areoptionally substituted {β-D-galactopyranosyl-(1→6)-}_(n)β-D-galactopyranosyl groups, with the proviso that “n” represents aninteger of 0 or over and, when both R₂ and R₈ are such glycosyl groups,the integers of “n” in each groups are independent each other. Exampleof the structural formula thereof is Chemical Formula 6 as shown inExperiment 4-4.

[0047] The forth more concrete example of the branchedcyclotetrasaccharides are those wherein R₄ and/or R₁₀ in Formula 1 areoptionally substituted{β-D-galactopyranosyl-(1→6)-}_(n)-D-galactopyranosyl groups, with theproviso that And represents an integer of 0 or over and, when both R₄and R₁₀ are such glycosyl groups, the integers of “n” in each groups areindependent each other. Examples of the structural formulae thereof areChemical Formulae 7 and 8 as shown in Experiment 4-5.

[0048] The fifth more concrete example of the branchedcyclotetrasaccharides are those wherein R₄ and/or R₁₀ in Formula 1 areoptionally substituted α-D-galactopyranosyl groups. Example of thestructural formula thereof is Chemical Formula 9 as shown in Experiment4-6.

[0049] The sixth more concrete example of the branchedcyclotetrasaccharides are those wherein R₂ and/or R₈ in Formula 1 areoptionally substituted β-D-chitosaminyl groups (“a chitosaminyl group”is also generally called “a glucosaminyl group”). Example of thestructural formula thereof is Chemical Formula 10 as shown in Experiment4-7.

[0050] Though the above examples of the branched cyclotetrasaccharidesof the present invention are respectively classified and exemplifiedbased on their constituent saccharides positioned at their branchedparts, the branched cyclotetrasaccharides may be those which have eitherone of these branched parts or two or more of them in an appropriatecombination. For example, a branched cyclotetrasaccharide whichcombinationally has both the structure of the branched part representedby the above first example, and the structure of the branched partrepresented by any of the above second to sixth examples. An example ofsuch structures is Chemical Formula 5 in Experiment 3-4.

[0051] As long as the branched cyclotetrasaccharides of the presentinvention have any of the above-mentioned structures, they should not berestricted to those which are produced by specific methods such asorganic syntheses and include those which are produced by enzymaticreactions. However, since the branched cyclotetrasaccharides areefficiently produced by the process according to the present inventiondescribed in detail below, those which are produced by the above processare advantageously used in a variety of fields. The branchedcyclotetrasaccharides of the present invention are provided in the formof a product consisting essentially of the branchedcyclotetrasaccharides as a saccharide component, usually, in a purifiedform with a purity of at least 90%, preferably, at least 95%, and morepreferably, at least 97%; in the form of a solution, amorphous powder,or molasses; or in the form of an isolated crystal. The branchedcyclotetrasaccharides in a crystal form can be isolated by crystallizingin water, an organic solvent such as lower alcohols anddimethylformamide, or a solvent in a mixture form of two or more of theabove solvents selected appropriately; and further separating theresulting crystals in a conventional manner. Crystals of branchedcyclotetrasaccharides in a hydrous or anhydrous form can be obtained bycrystallization in water. Examples of such hydrous crystal are those ofthe branched cyclotetrasaccharides represented by Chemical Formulae 1,2, 3, 6 and 7. These hydrous crystals can be converted into anhydrousones by heating at normal pressure or reduced pressure and at ambienttemperature. Crystals of these branched cyclotetrasaccharides can beidentified by conventional X-ray powder diffraction analysis.For-example, upon the analysis, the branched cyclotetrasaccharidesrepresented by Chemical Formulae 1, 2, 3, 6, and 7 have main diffractionangles (2θ) of (1) 8.1°, 12.2°, 14.2°, and 15.4°; (2) 5.6°, 8.8°, 16.9°,and 21.9°; (3)7.9°, 12.1°, 17.9°, and 20.2°; (4) 11.0°, 12.3°, 12.8°,and 24.9°; and (5) 8.7°, 13.0°, 21.7°, and 26.1°, respectively. Eachbranched cyclotetrasaccharide can be provided in the form of asaccharide composition comprising the same as a main ingredient. Thesaccharide composition, which contains one or more of the branchedcyclotetrasaccharides in an amount, usually, of at least 50%,preferably, at least 60%, more preferably, at least 70%, and morepreferably, at least 80% against the total amount of sugar components,on a dry solid basis (d.s.b.), is provided in the form of a solution,syrup, block, granule, crystalline powder containing hydrous and/oranhydrous crystal, amorphous crystalline powder, or molasses of thebranched cyclotetrasaccharide(s).

[0052] 2. Process for Producing Branched Cyclotetrasaccharides

[0053] The process for producing the branched cyclotetrasaccharides ofthe present invention employs the action of an enzyme that transfers aglycosyl group from a monosaccharide, oligosaccharide, or polysaccharideto cyclotetrasaccharide, and it is characterized in that it comprisesthe steps of allowing the enzyme to act on a mixture ofcyclotetrasaccharide and any of the above monosaccharide,oligosaccharide, and polysaccharide to form the desired branchedcyclotetrasaccharides, and collecting the produced branchedcyclotetrasaccharides.

[0054] 2.1. Preparation of Cyclotetrasaccharides

[0055] The process for producing cyclotetrasaccharide is notspecifically restricted. Examples of such are (1) a process forproducing cyclotetrasaccharide by contacting alternanase, i.e., ahydrolyzing enzyme, with alternan, i.e., a polysaccharide reported byGregory L. Cote et al. in European Journal of Biochemistry, Vol. 226,pp. 641-648 (1994); (2) a process for producing cyclotetrasaccharide bycontacting α-isomaltosyl-transferring enzyme withα-isomaltosylglucosaccharide; and (3) a process for producingcyclotetrasaccharide by contacting bothα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme with a saccharide, having a glucosepolymerization degree of at least two and the α-1,4 glucosyl bond as alinkage at its reducing end, such as a starch hydrolyzate. The terms“α-isomaltosylglucosaccharide-forming enzyme” and“α-isomaltosyl-transferring enzyme” as referred to as in the presentinvention mean enzymes having the following enzymatic activities (A) and(B), respectively, independently of their other enzymatic activities,not specified in (A) and (B), such as physicochemical properties andorigins.

[0056] (A) Acting on a saccharide, which has a glucose polymerizationdegree of “n” (“n” represents an integer of at least two) and the α-1,4glucosyl bond as a linkage at its non-reducing end, to form a saccharidehaving a glucose polymerization degree of “n+1” and having both theα-1,6 glucosyl bond as a linkage at its non-reducing end and the α-1,4glucosyl bond as a linkage other than the above linkage at itsnon-reducing end, without substantially increasing the reducing power ofthe saccharide.

[0057] (B) Acting on a saccharide, which has a glucose polymerizationdegree of at least three, the α-1,6 glucosyl bond as a linkage at itsnon-reducing end, and the α-1,4 glucosyl bond as a linkage other thanthe above linkage at its non-reducing end, to form cyclotetrasacchariderepresented bycyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}.

[0058] The formation mechanism of cyclotetrasaccharide by the aboveprocess (3) is roughly estimated as follows:

[0059] (I) α-isomaltosylglucosaccharide-forming enzyme acts on a glucoseresidue positioning at the reducing end of an α-1,4 glucan chain such asin glycogen or starch hydrolyzates, and intermolecularly transfers theglucose residue to the C-6 hydroxyl group of a glucose group positioningat the non-reducing end of another intact α-1,4 glucan chain, and toform an α-1,4 glucan chain having an α-isomaltosyl group at itsnon-reducing end.

[0060] (II) α-Isomaltosyl-transferring enzyme acts on the resultingα-1,4 glucan chain having an isomaltosyl group at its non-reducing end,and intermolecularly transfers the isomaltosyl group to the C-3 hydroxylgroup of a glucose group positioning at the non-reducing end of anotherintact α-1,4 glucan chain having an isomaltosyl group at itsnon-reducing end to form an α-1,4 glucan chain having anisomaltosyl-1,3-isomaltosyl group at its non-reducing end.

[0061] (III) Subsequently, α-isomaltosyl-transferring enzyme acts on theresulting α-1,4 glucan chain, having an isomaltosyl-1,3-isomaltosylgroup at its non-reducing end, to release theisomaltosyl-1,3-isomaltosyl group from the α-1,4 glucan chain via theintramolecular transferring action and to circularize the released groupfor forming cyclotetrasaccharide.

[0062] (IV) The resulting α-1,4 glucan chain is sequentially receivedthe above enzymatic reactions (I) to (III) to increase the yield ofcyclotetrasaccharides.

[0063] To produce cyclotetrasaccharide on an industrial scale, among theabove processes, the processes (2) and (3) are relatively advantageousin terms of its production cost and labors, particularly, the process(3) is more preferable. With reference to the above process (3) mainly,the process for producing cyclotetrasaccharide is explained below:

[0064] 2.1.1. α-isomaltosyl-Transferring Enzyme andα-isomaltosylglucosaccharide-Forming Enzyme

[0065] α-Isomaltosyl-transferring enzyme andα-isomaltosylglucosaccharide-forming enzyme can be obtained, forexample, by culturing a microorganism which produces either or both ofthe enzymes, and applying conventional methods for preparing enzymes tothe resulting culture. Bacillus globisporus C9 strain was deposited onApr. 25, 2000, with International Patent Organism Depositary NationalInstitute of Advanced Industrial Science and Technology Tsukuba Central6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken, Japan, and has beenmaintained therein under the accession number of FERM BP-7143; andBacillus globisporus C₁₋₁ strain was deposited on Apr. 25, 2000, withthe same depositary as above and has been maintained therein under theaccession number of FERM BP-7144, are particularly useful as sources ofthe above enzymes because they produce both the enzymes (the abovemicroorganisms may be called “Strain C9” and “Strain C11”, hereinafter).

[0066] The nutrient culture media and culture conditions used forculturing Strain C9, FERM BP-7143, and Strain C11, FERM BP-7144, are asfollows: Examples of the carbon sources usable in the present inventionare starches and phytoglycogens from plants; glycogens and pullulansfrom animals and microorganisms; partial hydrolyzates thereof;saccharides such as D-glucose, D-fructose, lactose, sucrose, mannitol,L-sorbitol, and molasses; and organic acids such as citric acid andsuccinic acid. The concentration of these carbon sources in nutrientculture media is appropriately changed depending on their types. Thenitrogen sources usable in the present invention are, for example,inorganic nitrogen-containing compounds such as ammonium salts andnitrates; and organic nitrogen-containing compounds such as urea, cornsteep liquor, casein, yeast extract, and beef extract. The inorganicingredients usable in the present invention are, for example, salts ofcalcium, magnesium, potassium, sodium, phosphoric acid., manganese,zinc, iron, copper, molybdenum, and cobalt. If necessary, amino acidsand vitamins can be appropriately used in combination.

[0067] Explaining the culture conditions, microorganisms are preferablycultured, usually, under aerobic conditions at temperatures, usually, of4 to 40° C., preferably, 20 to 37° C., at pHs, usually, of 4 to 10,preferably, 5 to 9, for 10 to 150 hours. The concentration of dissolvedoxygen (DO) can be controlled during the culturing, and it can be keptwithin the range of 0.5-20 ppm, for example, by means of controlling theaeration rate and the stirring speed, increasing or decreasing theoxygen concentration in gas used for aeration, and increasing ordecreasing the inner pressure of fermenters. The cultivation is freelycarried out batchwise, in a continuous manner, or in a semi-continuousmanner, as long as it affords the conditions in which microorganisms cangrow and produce α-isomaltosyl-transferring enzyme.

[0068] The cultures of Strain C9, FERM BP-7143, and Strain C11, FERMBP-7144, usually contain α-isomaltosyl-transferring enzyme andα-isomaltosylglucosaccharide-forming enzyme. Therefor, in the processfor producing cyclotetrasaccharide, the above cultures can be usedintact as an enzyme agent or used after purified into an enzyme agentwhich contains either or both of the above enzymes. For example, apartially purified enzyme agent, containing both the enzymes, can beobtained by removing cells from the cultures by means of a conventionalliquid-solid separation method, collecting the resulting supernatant,and optionally subjecting the supernatant to conventional methods forconcentrating proteins such as salting out using ammonium sulfate,sedimentation using acetone or alcohol, concentration in vacuo, andconcentration using membranes. If necessary, the enzyme agent thusobtain can be further treated by appropriately combining withconventional methods for purifying enzymes such as gel-filtrationchromatography, ion-exchange chromatography, affinity chromatography,and hydrophobic chromatography. From the separated fractions, those withthe desired enzymatic activities are separately collected into enzymeagents having either of the enzymes purified to the desired level.

[0069] The enzymatic activity of α-isomaltosyl-transferring enzyme canbe assayed as follows: Dissolve panose in 100 mm acetate buffer (pH 6.0)to give a concentration of 2% (w/v) f or a substrate solution, add 0.5ml of an enzyme solution to 0.5 ml of the substrate solution, and keepthe mixture at 35° C. for 30 min to proceed the formation ofcyclotetrasaccharide from panose. In the reaction system, glucose isformed together with cyclotetrasaccharide from panose. After thereaction, boil the reaction mixture for 10 min to suspend the reaction.Subject the resulting reaction mixture to the glucose oxidase method toquantify the glucose formed in the reaction mixture. In the presentinvention, one unit activity of α-isomaltosyl-transferring enzyme isdefined as the enzyme amount that forms one micromole of glucose perminute under the above enzymatic reaction conditions.

[0070] The α-isomaltosylglucosaccharide-forming enzyme activity can beassayed as follows: Add 0.5 ml of an enzymatic solution to 0.5 ml of asubstrate solution obtained by dissolving maltotriose in 100 mM acetatebuffer (pH 6.0) to give a concentration of 2% (w/v) is added, incubatethe mixture at 35° C. for 60 min to proceed the formation reaction ofisomaltosylmaltose from maltotriose. In the reaction system, maltose isformed together with isomaltosylmaltose from maltotriose. Thereafter,boil the reaction mixture for 10 min to suspend the enzymatic reaction.Subject the resulting mixture to-conventional HPLC for detecting andquantifying maltose formed in the reaction mixture to quantify themaltose. In the present invention, one unit activity of theα-isomaltosylglucosaccharide-forming enzyme is defined as the enzymeamount that forms one micromole of maltose per minute under the aboveenzymatic reaction conditions.

[0071] Table 1 shows the physicochemical properties of both the enzymesobtained from Strain C9, FERM BP-7143; and Strain C11, FERM BP-7144,which are confirmed-by the index in the above defined enzymaticactivities. TABLE 1 α-Isomaltosyl-transferringα-Isomaltosylglucosaccharide- enzyme forming enzyme Molecular weightAbout 82,000 to about 132,000 daltons About 117,000 to about 160,000altons (Analysis method) (SDS-PAGE) (SDS-PAGE) Isoelectric point pI ofabout 5.0 to about 6.0 pI of about 4.7 to about 5.7 (Analysis method)(Isoelectrophoresis using ampholine) (Isoelectrophoresis using mpholine)Optimum temperature About 45° C. to about 50° C. About 40° C. to about45° C. (Analysis condition) (Reaction at pH 6.0 for 30 min) (Reaction atpH 6.0 for 60 min) About 45° C. to about 50° C. (Reaction under the samecondition as above in the presence of 1 mM Ca²⁺) Optimum pH pH of about5.5 to about 6.0 pH of about 6.0 to about 6.5 (Analysis condition)(Reaction at 35° C. for 30 min) (Reaction at 35° C. for 60 min) Thermalstability About 40° C. or lower About 35° C. to about 40° C. or lower(Analysis condition) (Reaction at pH 6.0 for 60 min) (Reaction at pH 6.0for 60 min) About 40° C. to about 45° C. or lower (Reaction under thesame condition as above in the presence of 1 mM Ca²⁺) pH Stability pH ofabout 4.0 to about 9.0 pH of about 4.5 to about 10.0 (Analysiscondition) (Incubated at 4° C. for 24 hours) (Incubated at 4° C. for 24hours)

[0072] When an enzyme agent is a composition ofα-isomaltosyl-transferring enzyme andα-isomaltosylglucosaccharide-forming enzyme, it has an activity offorming cyclotetrasaccharide from partial starch hydrolyzates. Theactivity of forming cyclotetrasaccharide from partial starchhydrolyzates (the term “a cyclotetrasaccharide forming activity” meansthe above activity, hereinafter) can be assayed as follows: Add 0.5 mlof an enzyme solution to 0.5 ml of a substrate solution obtained bydissolving “PINE-DEX #100™”, a partial starch hydrolyzate commercializedby Matsutani Chemical Ind., Tokyo, Japan, in 50 mM acetate buffer (pH6.0) to give a concentration of 2% (w/v), incubate the mixture at 35° C.for 60 min to proceed the formation reaction ofisomaltosylglucosaccharide-forming enzyme can be also obtained by therecombinant DNA technology. The same applicant as the present inventiondisclosed the nucleotide sequences of DNAs which encode theα-isomaltosyl-transferring enzyme andα-isomaltosylglucosaccharide-forming enzyme from Strain C11, FERMBP-7444, in Japanese Patent Application Nos. 350,142/2000 and5,441/2001. As disclosed in these specifications, each of the abovenucleotide sequences includes a nucleotide sequence of a coding region,which corresponds to a precursor of each of the enzymes having a signalpeptide at the N-terminus, and those in the 5′- and 3′-non-translationalregions. In the sequence listing of the present specification, SEQ IDNO:1 represents a nucleotide sequence of a coding region correspondingto a precursor of α-isomaltosyl-transferring enzyme from Strain C11disclosed in Japanese Patent Application No. 350,142/2000, and SEQ IDNO:2 represents a nucleotide sequence of a coding region correspondingto a precursor of α-isomaltosylglucosaccharide-forming enzyme fromStrain C11 disclosed in Japanese Patent Application No. 5,441/2001. Withreference to these nucleotide sequences, both of the above enzymes canbe prepared by using conventional recombinant DNA technologies such as aDNA cloning method, site-directed mutagenesis, transformation ofmicroorganisms, and artificial expression method of DNAs. Conventionalrecombinant DNA technologies are, for example, described in detail in“Molecular cloning, A LABORATORY MANUAL, THIRD EDITION” by J. Sambrook,published by Cold Spring Harbor Laboratory Press, 2001.

[0073] 2.1.2. Preparation of Cyclotetrasaccharide Usingα-Isomaltosyl-Transferring Enzyme andα-Isomaltosylglucosaccharide-Forming Enzyme

[0074] In preparing cyclotetrasaccharide from a substrate such aspartial starch hydrolyzates by using α-isomaltosyl-transferring enzymeand α-isomaltosylglucosaccharide-forming enzyme, the former enzyme isallowed to act on a substrate, usually, in a aqueous solution form,after the action of or under the coaction of the latter enzyme. Thecoaction of the above enzymes is relatively preferable in view of theproduction efficiency of cyclotetrasaccharide. Examples of thesubstrates used in this method are saccharides having a glucosepolymerization degree of at least two and the α-1,4 glucosyl bond as alinkage at their non-reducing ends; maltooligosaccharides,maltodextrins, amylodextrins, amyloses, amylopectins, soluble starches,liquefied starches, gelatinized starches, and glycogens. Considering theproduction cost, terrestoreal starches such as corn, wheat, and rice;and subterranean starches such as potato, sweet potato, and tapioca arepreferably used as starting materials. Both of the enzymes arepreferably allowed to act on liquefied starches prepared by allowingliquefying-type amylases to act on suspensions of the above starches orheating the suspensions under acid conditions. The lower the “DE”(dextrose equivalent) of liquefied starch, the higher the yield ofcyclotetrasaccharide becomes. The DE is usually 20 or lower, preferably,12 or lower, and more preferably, five or lower. Prior to or in parallelwith the action of α-isomaltosyl-transferring enzyme andα-isomaltosylglucosaccharide-forming enzyme on liquefied starch,debranching enzymes such as pullulanase or isoamylase can beadvantageously used because they may increase the yield ofcyclotetrasaccharide.

[0075] The concentration of substrates is not specifically restricted aslong as cyclotetrasaccharide is formed. The higher the concentration,the higher the yield of cyclotetrasaccharide per batch becomes; usually,it is 0.1%(w/w) or higher, preferably, one percent (w/w) or higher,d.s.b. Though substrates can be used in a solution form with aconcentration over their water solubility, the concentration ispreferably set to 40% (w/w) or lower, preferably, 35% (w/w) or lower,d.s.b., to ease handlings.

[0076] The reaction condition is not specifically restricted as along ascyclotetrasaccharide is formed. For example, any temperatures of fromambient temperature to 50° C., preferably, 30° C. to 45° C., can besuitably used; and any pHs of from 4.5 to 8, preferably, from 5.5 to 7can be preferably used. Met-al ions such as Ca²⁺ and Mg²⁺, whichstabilize any of the enzymes used, can be advantageously coexisted in areaction mixture. The reaction time can be arbitrarily set in view ofthe reaction progress, depending on the amount of the enzymes used.

[0077] If necessary, other saccharide transferring enzymes can beadvantageously used when α-isomaltosyl-transferring enzyme andα-isomaltosylglucosaccharide-forming enzyme are allowed to act on asubstrate. For example, the combination use of cyclomaltodextringlucanotransferase may increase the production yield ofcyclotetrasaccharide as compared with the case without the combinationuse.

[0078] As an enzyme agent used as α-isomaltosyl-transferring enzyme andα-isomaltosylglucosaccharide-forming enzyme to be allowed to act on asubstrate, any microorganism which produces both the enzymes can beused. To use microorganisms as such an enzyme agent, for example, StrainC9, FERM BP-7143, and Strain C11, FERM BP-7144, which are capable offorming both the enzymes, are cultured and proliferated up to reach thedesired cell density under their proliferation conditions. As theculture conditions for the above microorganisms, the above ones forforming enzymes can be arbitrarily used. The resulting cultures can beallowed to act on substrates similarly as in the above enzyme agent.

[0079] The reaction mixtures thus obtained contain cyclotetrasaccharideand they can be used intact as cyclotetrasaccharide solutions or usedafter purification. To purify cyclotetrasaccharide, conventionalpurification methods for saccharides can be appropriately employed.Examples of such are decoloration with activated charcoal; desaltingwith ion-exchange resins in H- and OH-forms; fractionation by columnchromatography using an ion-exchange resin, activated charcoal, andsilica gel (usually called “chromatography”); separation sedimentationusing organic solvents such as alcohol and acetone; separation usingmembranes with appropriate separability; and decomposition and treatmentto remove coexisting or remaining other saccharides, for example,enzymatic treatment with amylases such as α-amylase, β-amylase, andglucoamylase, and α-glucosidase, fermentation treatment with yeasts, andalkaline treatment. An appropriate combination use of the abovepurification methods advantageously increase the purity ofcyclotetrasaccharide. The resulting purified cyclotetrasaccharide andsaccharide compositions containing the same can be prepared into thedesired form of a solution, syrup, block, powder, granule, crystal,etc., by applying thereunto an appropriate combination of treatmentssuch as concentration, crystallization, drying, pulverization, anddissolution.

[0080] The above outlines the process for producingcyclotetrasaccharide, which uses α-isomaltosyl-transferring enzyme andα-isomaltosylglucosaccharide-forming enzyme in combination. In themethod of using only α-isomaltosyl-transferring enzyme (theabove-mentioned process for producing cyclotetrasaccharide (2)), such anenzyme is prepared similarly as above and then allowed to act onα-isomaltosylglucosaccharide such as panose commercialized or preparedin a usual manner, and optionally the formed cyclotetrasaccharide can befurther purified similarly as above.

[0081] 2.2. Enzyme Which Transfers Glycosyl Group toCyclotetrasaccharide

[0082] Any enzymes can be used in the process of the present inventionindependently of their unrequisite actions and origins other than theability of forming the above-identified branched cyclotetrasaccharides,represented by Formula 1, by transferring a glycosyl group frommonosaccharides, oligosaccharides, or polysaccharides (hereinafter,these saccharides are called “donors of glycosyl group”). Examples ofsuch enzymes are cyclomaltodextrin glucanotransferase,α-isomaltosyl-transferring enzyme, α-isomaltosylglucosaccharide-formingenzyme, β-galactosidase, α-galactosidase, and lysozyme. In terms of thetransferring of a glycosyl group to cyclotetrasaccharide (the expressionof “transfer of glycosyl group” may be abbreviated as “glycosyltransfer”, hereinafter), the properties of each enzymes are brieflyexplained in the below:

[0083] Cyclomaltodextrin glucanotransferase (EC 2.4.1.19) usuallytransfers a glycosyl group from, as a donor of glycosyl group, asaccharide having the α-1,4 glucosyl bond as a linkage and having aglucose polymerization degree of at least two, such asmaltooligosaccharide, maltodextrin, amylodextrin, amylose, amylopectin,soluble starch, liquefied starch, gelatinized starch, or glycogen; andusually forms the branched cyclotetrasaccharides as in the first exampleof the aforesaid paragraph “1. Branched cyclotetrasaccharides”.

[0084] α-Isomaltosyl-transferring enzyme transfers a glycosyl groupfrom, as a donor of glycosyl group, α-isomaltosylglucosaccharides suchas panose to cyclotetrasaccharide and usually forms the branchedcyclotetrasaccharides as in the second example of the aforesaidparagraph “1. Branched cyclotetrasaccharides”, particularly, thoserepresented by Chemical formulae 3 and 4.

[0085] α-Isomaltosylglucosaccharide-forming enzyme usually formssaccharides, which have the α-1,4 glucosyl bond as a linkage and aglucose polymerization degree of at least three such asmaltooligosaccharide, maltodextrin, amylodextrin, amylose, amylopectin,soluble starch, liquefied starch, gelatinized starch, or glycogen; andusually forms the branched cyclotetrasaccharides represented by Formula1.

[0086] β-Galactosidase (EC 3.2.1.23) usually transfers a glucosyl groupfrom lactose as a donor of glycosyl group to cyclotetrasaccharide andusually forms the branched cyclotetrasaccharides as in the third andforth examples of the above paragraph “1. Branchedcyclotetrasaccharides”. Depending on the origin of β-galactosidase used,the type or the composition of the formed branched cyclotetrasaccharidesmay differ. For example, the enzymes from the microorganisms of thespecies Bacillus circulans relatively efficiently form the branchedcyclotetrasaccharides represented by Chemical Formula 6, while thosefrom the microorganisms of the species Aspergillus niger form thebranched cyclotetrasaccharides represented by Chemical Formulae 6 to 8.

[0087] α-Galactosidase (EC 3.2.1.22) usually transfers a glycosyl groupfrom melibiose as a donor of glycosyl group to cycloterasaccharide andusually forms branched cyclotetrasaccharides, particularly, thoserepresented by Chemical Formula 9 as in the fifth example of the aboveparagraph “1. Branched cyclotetrasaccharides”.

[0088] Lysozyme (EC 3.2.1.17) usually transfers to cyclotetrasaccharidea glycosyl group from oligosaccharides or polysaccharides, as a donor ofa glycosyl group, such as N-acetylchitooligosaccharide and chitin, whichare composed of N-acetylchitosamine (also known as N-acetylglucosamine)as a constituent saccharide and the β-1,4 glycosyl bond; and usuallyforms the branched cyclotetrasaccharides, particularly, thoserepresented by Chemical Formula 10 as in the sixth example of the aboveparagraph “1. Branched cyclotetrasaccharides”.

[0089] The above describes the transferring reactions that are mainlycatalyzed by the above enzymes when they each independently act oncyclotetrasaccharide, however, combination use of two or more of themcan form other branched cyclotetrasaccharides. For example, whencyclomaltodextrin glucanotransferase and α-isomaltosyl-transferringenzyme are combinationally allowed to act on both cyclotetrasaccharideand an appropriate saccharide as a donor of glycosyl group, the branchedcyclotetrasaccharides represented by Chemical Formula 5 will be formed.Another combination of the above enzymes may form different types ofbranched cyclotetrasaccharides. In addition to the above exemplifiedenzymes, saccharide-related enzymes such as kojibiose phosphorylase,disclosed in Japanese Patent Kokai No. 304,882/98 applied for by thesame applicant as the present applicant, glycogen phosphorylase (EC2.4.1.1), maltose phosphorylase (EC 2.4.1.8), α-glucosidase (EC3.2.1.20), oligo-1,6-glucosidase (EC 3.2.1.10), and β-glucosidase (EC3.2.1.21) can be arbitrarily used in the process of the presentinvention as long as they transfer a glycosyl group from its donor tocyclotetrasaccharide and form the branched cyclotetrasaccharidesrepresented by the above-mentioned Formula 1. Particularly, the abovekojibiose phosphorylase can be advantageously used to meet its purposebecause, depending on reaction conditions, it can transfer a glycosylgroup from either glucose-1-phosphate, as a donor saccharide, or aglycosyl group to cyclotetrasaccharide and form the branchedcyclotetrasaccharides represented by Formula 1, with the proviso thatone or more of R₃, R₆, R₉, and R₁₂ are oligoglucosyl groups havingα-1,2-glucosyl bonds such as a glycosyl group and a kojibiosyl group.

[0090] All the above-exemplified enzymes are well known in the art andany of commercialized preparations thereof or those prepared byconventional reports on their preparations can be used in practicing thepresent invention.

[0091] 2.3. Preparation of Branched Cyclotetrasaccharides fromCyclotetrasaccharide

[0092] To produce the branched cyclotetrasaccharides of the presentinvention, an enzyme (hereinafter abbreviated as “aglycosyl-transferring enzyme” in this paragraph 2.3.) is firstlyselected depending on the structure of the aimed branchedcyclotetrasaccharides, and is obtained by purchasing a commercializedenzyme preparation of such an enzyme or by preparing the enzyme in aconventional manner. A suitable saccharide as a donor of glycosyl groupis obtained by purchasing a commercialized product thereof or bypreparing the donor in a conventional manner, depending on theproperties of the enzymes used. The cyclotetrasaccharide used in thepresent invention is prepared according to any of the methods describedin the above paragraph 2.1.

[0093] Using a glycosyl-transferring enzyme and any of theabove-mentioned substrates, i.e., cyclotetrasaccharide and a donor ofglycosyl group, a mixture of substrates usually in the form of anaqueous solution is provided and then admixed with the enzyme to effectenzymatic reaction for forming the desired branchedcyclotetrasaccharides in the resulting reaction mixture. The reactionconditions for the glycosyl-transferring enzyme are not specificallyrestricted as long as the desired branched cyclotetrasaccharides areformed. In practicing the enzymatic reaction in an aqueous system,though it varies depending on the water solubilities of substrates usedat their reaction temperatures, the concentrations of substrates, i.e.,the one of cyclotetrasaccharide is usually set to 1 to 40% (w/w),preferably, 5 to 35% (w/w); and the one of the donor of glycosyl groupis set to the highest possible level but within the range that the donordissolves therein, usually, in an amount of at least a half time of,preferably, at least the same as, and more preferably, at least twotimes of that of cyclotetrasaccharide. The reaction temperatures and pHsare arbitrarily selected in view of the enzymological properties of theglycosyl-transferring enzymes used as long as they do not completelyinactivate the enzymes during their enzymatic reactions. The amount ofenzymes is arbitrarily selected to yield the desired product at the endof enzymatic reaction depending on the substrate concentration and thereaction time used.

[0094] Though the resulting reaction mixture with branchedcyclotetrasaccharides can be used intact as a saccharide compositioncontaining the same, it is usually purified-from the mixture before use.The branched cyclotetrasaccharides are purified according toconventional methods: Decoloration with an activated charcoal anddesalting with ion-exchange resins in H- and OH-forms can be arbitrarilyused. If necessary, the degree of purification of the desired branchedcyclotetrasaccharides can be advantageously increased by an appropriatecombination of fractionation by column chromatography using ion-exchangeresins, activated cachols, and silica gels; fractional precipitationusing organic solvents such as alcohol and acetone; separation usingmembranes with an adequate separability; and other treatments ofdecomposing and removing the coexisting or remaining other saccharides,for example, enzymatic treatments with amylases such α-amylase,β-amylase, and glucoamylase, and α-glucosidase, fermentations withyeasts, and alkaline treatments. The resulting purified branchedcyclotetrasaccharide(s) and saccharide compositions containing the samecan be treated with an appropriate combination of treatments such asconcentration, crystallization, drying, pulverization, and dissolution,and optionally mixed with an appropriate saccharide other than thebranched cyclotetrasaccharides of the present invention into the desiredproducts in the form of a solution, syrup, block, crystalline powdercontaining hydrous- and/or anhydrous-crystals, amorphous powder,granule, isolated crystal, or molasses.

[0095] In the reaction to form cyclotetrasaccharide via the action ofα-isomaltosyl-transferring enzyme andα-isomaltosylglucosaccharide-forming enzyme, or the action ofα-isomaltosyl-transferring enzyme on α-isomaltosyl glucosaccharide, asshown in the above paragraph 2.1.2., the branched cyclotetrasaccharidesof the present invention are formed as by-products in different yieldsdepending on the reaction conditions used. Among the branchedcyclotetrasaccharides, main components are those represented by ChemicalFormula 1, and those in the second example of the above paragraph “1.Branched cyclotetrasaccharides”, and those of Chemical Formulae 3, 4,and 5. Therefore, to meet the object, the branched cyclotetrasaccharidesof the present invention can be obtained by separating fromcyclotetrasaccharide in their reaction mixture obtained after the actionof the aforesaid both enzymes or the sole use ofα-isomaltosyl-transferring enzyme.

[0096] 3. Use of Branched Cyclotetrasaccharides

[0097] Since having a common basic structure, the branchedcyclotetrasaccharides of the present invention usually havesubstantially the same properties and functions as ofcyclotetrasaccharide. Therefore, the branched cyclotetrasaccharides canbe used for purposes similarly as in cyclotetrasaccharide. The followingis a brief description of the uses of the branched cyclotetrasaccharidesof the present invention which can be used in accordance with those ofthe cyclotetrasaccharide disclosed in Japanese Patent Application No.234,937/2000 (International Publication No. WO 02/10,361 A1).

[0098] The branched cyclotetrasaccharides of the present invention areusually stable, non-reducing saccharides, which have a white powder formand a relatively low- or non-sweetness, refined taste. When mixed andprocessed with other materials, particularly, amino acids or materialscontaining amino acids such as oligopeptides and proteins, the branchedcyclotetrasaccharides hardly induce the browning reaction, hardly-causeundesirable smell, and hardly spoil the other materials mixed. Thus, thebranched cyclotetrasaccharides of the present invention can beincorporated and used as materials or bases in many fields such as foodproducts, cosmetics, and pharmaceuticals.

[0099] Since the branched cyclotetrasaccharides of the present inventionhave an inclusion ability, they effectively inhibit the volatilizationand the deterioration of flavoring ingredients and effectiveingredients, and quite satisfactorily, stably keep these ingredients. Inthis case, if necessary, the above stabilization by inclusion can beenhanced by the combination use of other cyclotetrasaccharides such ascyclodextrins, branched cyclodextrins, cyclodextrans, and cyclofractans.The cyclotetrasaccharides such as cyclodextrins should not be limited tothose with the highest purity, and include those with a lower purity,for example, partial starch hydrolyzates rich in maltodextrins alongwith cyclodextrins can be arbitrarily used.

[0100] Since cyclotetrasaccharide is not hydrolyzed by amylase orα-glucosidase, it is not assimilated and absorbed by living bodies whenorally taken, hardly fermented by intestinal bacteria, and utilized asan aqueous dietary fiber with quite low calories. Also, sincecyclotetrasaccharide is hardly assimilated by dental-caries-inducingbacteria, it can be used as a sweetener which does not substantiallycause dental caries. Further, cyclotetrasaccharide also has a functionof preventing the adhesion and solidification of solid materials withinthe oral cavity. The branched cyclotetrasaccharides of the presentinvention have the same basic structure as cyclotetrasaccharide and havea high utility as saccharides with a relatively low calorie andcariogenicity compared with conventional saccharides susceptible tofermentation by dental-caries inducing bacteria. The branchedcyclotetrasaccharides of the present invention are non-poisonous,harmless saccharides, free from side effect, and useful as stablematerials or bases, and when they are in the form of a crystallineproduct, they can be arbitrarily processed into tablets or sugar coatedtablets in combination with binders such as pullulan, hydroxyethylstarch, or poly(vinylpyrrolidone). The branched cyclotetrasaccharides ofthe present invention have properties such as osmoticpressure-controlling ability, filling ability, gloss-imparting ability,moisture-retaining ability, viscosity-imparting ability, crystallizationpreventing ability of other saccharides, and insubstantialfermentability. Therefore, the branched cyclotetrasaccharides and thesaccharide compositions comprising the same can be advantageously usedin compositions such as food products, articles of taste includingtobaccos and cigarettes, feeds, baits, cosmetics, and pharmaceuticals assaccharide seasonings, taste-improving agents, quality-improving agents,stabilizers, color-deterioration preventing agents, and fillers.

[0101] The branched cyclotetrasaccharides and the saccharidecompositions comprising the same can be used intact as seasonings togive a refined taste, and if necessary, they can be used in combinationwith other sweeteners such as a powdered starch hydrogenate, glucose,isomerized sugar, sugar, maltose, trehalose, honey, maple sugar,sorbitol, maltitol, dihydrochalcones, stevioside, α-glycosylstevioside,extract from Momordica grosvenori, glycyrrhizin, thaumatin,L-aspartylphenylalanine methylester, saccharin, acesulfam K, sucralose,glycine, and alanine; or fillers such as dextrins, starches, andlactose. Particularly, the branched cyclotetrasaccharide and thesaccharide compositions comprising the same can be preferably used aslow-caloric or diet sweeteners in combination with one or moresweeteners such as meso-erythritol, xylitol, or maltitol; sweetenerswith high sweetness such as α-glycosylstevioside, thaumatin,L-aspartylphenylalanine methylester, saccharin, acesulfam K, andsucralose.

[0102] The branched cyclotetrasaccharides and the saccharidecompositions comprising the same can be arbitrarily used intact oroptionally mixed with fillers, excipients, and binders; and then shapedinto appropriate forms such as a granule, sphere, short stick/rod,sheet, cube, and tablet.

[0103] The refined taste of the branched cyclotetrasaccharides and thesaccharide compositions comprising the same well harmonize with othermaterials having sour-, acid-, salty-, delicious-, astringent-, andbitter-tastes; and have a satisfactorily-high acid- and heat-tolerance.Thus, they can be favorably used as sweeteners, taste-improving agents,quality-improving agents, etc., in seasonings, for example, a soy sauce,powdered soy sauce, miso, “funmatsu-miso” (a powdered miso), “morom-i”(a refined sake), “hishio” (a refined soy sauce), “furikake” (a seasonedfish meal), mayonnaise, dressing, vinegar, “sanbai-zu” (a sauce ofsugar, soy sauce and vinegar), “funmatsu-sushi-su” (powdered vinegar forsushi), “chuka-no-moto” (an instant mix for Chinese dish), “tentsuyu” (asauce for Japanese deep-fat fried food), “mentsuyu” (a sauce forJapanese vermicelli), sauce, catsup, “yakiniku-no-tare” (a sauce forJapanese grilled meat), curry roux, instant stew mix, instant soup mix,“dashi-no-moto” (an instant stock mix), mixed seasoning, “mirin” (asweet sake), “shin-mirin” (a synthetic mirin), table sugar, and coffeesugar. Also, the cyclotetrasaccharide and the saccharide compositionscomprising the same can be arbitrarily used to sweeten and improve thetaste and quality of “wagashi” (Japanese cakes) such as “senbei” (a ricecracker), “arare” (a rice cake cube), “okoshi” (a millet-and-rice cake),“gyuhi” (a starch paste), “mochi” (a rice paste) and the like, “manju”(a bun with a bean-jam), “uiro” (a sweet rice jelly), “an” (a bean jam)and the like, “yokan” (a sweet jelly of beans), “mizu-yokan” (a softadzuki-bean jelly), “kingyoku” (a kind of yokan), jelly, pao deCastella, and “amedama” (a Japanese toffee); Western confectioneriessuch as a bun, biscuit, cracker, cookie, pie, pudding, butter cream,custard cream, cream puff, waffle, sponge cake, doughnut, chocolate,chewing gum, caramel, nougat, and candy; frozen desserts such as an icecream and sherbet; processed foods of fruit and vegetables such as ajam, marmalade, and “toka” (conserves); pickles and pickled productssuch as a “fukujin-zuke” (red colored radish pickles), (ettara-ue (akind of whole fresh radish pickles), “senmai-zuke” (a kind of slicedfresh radish pickles), and “rakkyo-zuke” (pickled shallots); premixesfor pickles and pickled products such as a “takuan-zuke-no-moto” (apremix for pickled radish), and “hakusai-zuke-no-moto” (a premix forfresh white rape pickles); meat products such as a ham and sausage; fishmeat products such as a fish ham, fish sausage, “kamaboko” (a steamedfish paste), “chikuvwa” (a kind of fish paste), and “tenpura” (aJapanese deep-fat fried fish paste); “chinm-i” (relish) such as a“uni-no-shiokara” (salted guts of sea urchin), “ika-no-shiokara” (saltedguts of squid), “su-konbu” (processed tangle), “saki-surume” (driedsquid strips), “-fugu-no-mirin-boshi” (a dried mirin-seasonedswellfish), seasoned fish flour such as of Pacific cod, sea bream,shrimp, etc; “tsukudani” (foods boiled down in soy sauce) such as thoseof layers, edible wild plants, dried squids, small fishes, andshellfishes; daily dishes such as a “nimame” (cooked beans), potatosalad, and “konbu-maki” (a tangle roll); milk products; canned andbottled products such as those of meat, fish meat, fruit, and vegetable;alcoholic beverages such as a synthetic sake, fermented liquor, sake,fruit wine, sparkling alcoholic beverage, beer; soft drinks such as acoffee, cocoa, juice, carbonated beverage, lactic acid beverage, andbeverage with lactic acid bacteria; instant food products such asinstant pudding mix, instant hot cake mix, instant juice or soft drink,instant coffee, “sokuseki-shjiuko” (an instant mix of adzuki-bean soupwith rice cake), and instant soup mix; and other foods and beveragessuch as a food for babies, food for therapy, health/tonic drink, peptidefood, and frozen food. The branched cyclotetrasaccharides and thesaccharide compositions comprising the same can be arbitrarily used toimprove the taste preference and property of feeds and pet foods foranimals and pets such as domestic animals, poultry, honey bees, silkwarms, and fishes; and also they can be arbitrary used as a tastepreference-improving agent, taste-improving agent, flavor-impartingagent, quality-improving agent, and stabilizer in other tastableproducts, cosmetics, and pharmaceuticals in a solid, paste, or liquidform such as a tobacco, cigarette, tooth paste, lipstick, rouge,lipcream, internal liquidmedicine, tablet, troche, cod liver oil drop,cachou, oral refreshment, and gargle. When used as a quality-improvingagent or stabilizer, the branched cyclotetrasaccharides and thesaccharide compositions comprising the same can be arbitrarily used inbiologically active substances susceptible to lose their activeingredients, as well as health foods and pharmaceuticals containing suchbiologically active substances. Examples of such biologically activesubstances are liquid preparations containing lymphokines such as α-, β-and γ-interferons, tumor necrosis factor-α (TNF-α), tumor necrosisfactor-β (TNF-β), macrophage migration inhibitory factor,colony-stimulating factor, transfer factor, and interleukin 2; liquidpreparations containing hormones such as insulin, growth hormone,prolactin, erythropoietin, and follicle-stimulating hormone; liquidpreparations containing biologically active substaces such as BCGvaccine, Japanese encephalitis vaccine, measles vaccine, live poliovaccine, smallpox vaccine, tetanus toxoid, Trimeresurus antitoxin, andhuman immunoglobulin; liquid preparations containing antibiotics such aspenicillin, erythromycin, chloramphenicol, tetracycline, streptomycin,and kanamycin sulfate; liquid preparations containing vitamins such asthiamine, riboflavin, L-ascorbic acid, cod liver oil, carotenoid,ergosterol, and tocopherol; liquid preparation containing highlyunsaturated fatty acids and ester derivatives thereof such as EPA, DHA,and arachidonic acid, or containing enzymes such as lipase, elastase,urokinase, protease, β-amylase, isoamylase, glucanase, and lactase;extracts such as ginseng extract, snapping turtle extract, chlorellaextract, aloe extract, and propolis extract; biologically activesubstances such royal jelly; and pastes with alive viruses, lactic acidbacteria or yeasts. By applying to the above biologically activesubstances, the branched cyclotetrasaccharides and the saccharidecompositions containing the same to the above biologically activesubstances facilitate the production of stabilized, high-quality healthfoods and pharmaceuticals in the form of a liquid, paste, or solid withless fear of losing or inactivating the effective ingredients andactivities of the substances.

[0104] The methods for incorporating the branched cyclotetrasaccharidesor the saccharide compositions containing the same into the aforesaidcompositions are those which can incorporate them into a variety ofcompositions before completion of their processings. For such purposes,the following conventional methods such as mixing, kneading, dissolving,melting, soaking, penetrating, dispersing, applying, coating, spraying,injecting, crystallizing, and solidifying can be appropriately selected.The percentage of the branched cyclotetrasaccharides or the saccharidecompositions containing the same to the final compositions is usually atleast 0.1%, desirably, at least 1%, d.s.b. in addition to the above usessimilar to those for cyclotetrasaccharide, the branchedcyclotetrasaccharides of the present invention may exhibit an activityof growing bifid bacteria and it can be used as a factor for suchpurpose when incorporated into foods, beverages, health foods, healthfood supplements, and pharmaceuticals. In this case, other saccharides,having an activity of growing bifid bacteria, such as lactosucrose(4-β-D-galactosylsucrose), N-acetyl-D-chitosamine(N-acetyl-D-glucosamine), and lactulose can be advantageously usedtogether. Since the branched cyclotetrasaccharides of the presentinvention has a function of preventing crystallization ofcyclotetrasaccharide in an aqueous solution, it can be used as acrystallization-preventing agent for such purpose. For example, when thebranched cyclotetrasaccharides or the saccharide compositions containingthe same of the present invention are added to an aqueous solution,containing cyclotetrasaccharide alone or in combination with anotherreducing saccharide(s) such as glucose, maltose and panose, obtained byany of the above methods, the resulting solution can be concentratedinto a supersaturated solution of cyclotetrasaccharide. Further, theabove solution can be used after hydrogenation by reducing saccharidescontained therein into sugar alcohols, if necessary. The highcyclotetrasaccharide content solutions thus obtained can be moreefficiently stored in tanks and transported by pumps and tank lorriescompared with solutions free of the branched cyclotetrasaccharides ofthe present invention. Thus, the branched cyclotetrasaccharides of thepresent invention are remarkably useful in industrial handlings ofcyclotetrasaccharide.

[0105] The following experiments explain the present invention in moredetail:

EXPERIMENT 1 Isolation and Identification of Cyclotetrasaccharides fromCulture of Microorganism

[0106] A liquid culture medium, consisting of 5% (w/v) of panose, 1.5%(w/v) of “ASAHIMEAST™”, a yeast extract commercialized by AsahiBreweries, Ltd., Tokyo, Japan, 0.1% (w/v) of dipotassium phosphate,0.06% (w/v) of sodium dihydrogen phosphate dodecahydrate, 0.05% (w/v)magnesium sulfate heptahydrate, and water was placed in a 500-mlErlenmeyer flask in an amount of 100 ml, was sterilized by autoclavingat 121° C. for 20 min, cooled, and then seeded with a seed culture ofBacillus globisporus C9 strain, FERM BP-7143, followed by culturingunder rotary-shaking conditions at 27° C. and 230 rpm for 48 hours andcentrifuging the resulting culture to remove cells to obtain asupernatant. The supernatant was autoclaved at 120° C. for 15 min andthen cooled, and the resulting insoluble substances were removed bycentrifugation to obtain a supernatant. A non-reducing saccharide, whichwas positive on the sulfuric acid-methanol method and negative on thediphenylamine-aniline method, was observed in this supernatant.

[0107] About 90 ml of the supernatant after the autoclaving was adjustedto pH 5.0 and 45° C. and then incubated for 24 hours after the additionof 1,500 units per gram of solids of “TRANSGLUCOSIDASE L AMANO™”, anα-glucosidase commercialized by Amano Pharmaceutical Co., Ltd., Aichi,Japan, and 75 units per gram of solids of “GLUCOTEAM™”, a glucoamylasecommercialized by Nagase Biochemicals, Ltd., Kyoto, Japan. Thereafter,the resulting mixture was adjusted to pH 12-by the addition of sodiumhydroxide and boiled after two hours to decompose the reducing sugars inthe supernatant. After removing insoluble substances by filtration, thefiltrate was decolored and desalted with “DIAION PK218™” and “DIAIONWA30™”, ion-exchange resins commercialized by Mitsubishi ChemicalIndustries, Ltd., Tokyo, Japan, and further desalted with “DIAIONSK-1B™”, a cation exchange resin commercialized by Mitsubishi ChemicalIndustries, Ltd., Tokyo, Japan, and “AMBERLITE IRA411™”, an anionexchange resin commercialized by Japan Organo-Co., Ltd., Tokyo, Japan,followed by filtrating with a membrane, concentrated by an evaporator,and lyophilized in vacuo to obtain about 0.5 g solid of a non-reducingsaccharide powder.

[0108] The purity of the non-reducing saccharide powder was analyzed onhigh-performance liquid chromatography (abbreviated as “HPLC”hereinafter). HPLC was carried out using “SHOWDEX KS-801 column™”, ShowaDenko K.K., Tokyo, Japan, at a column temperature of 60° C. and a flowrate of 0.5 ml/min of water, and using “RI-8012™”, a differentialrefractometer commercialized by Tosoh Corporation, Tokyo, Japan. Asshown in FIG. 1, a single peak was detected at an elution time of 10.84min, revealing that the saccharide had a high purity of at least 99.9%.

[0109] Mass analysis by fast atom bombardment mass spectrometry (called“FAB-MS”) of the non-reducing saccharide, obtained by the aforesaidmethod, significantly detected a proton-addition-molecular ion with amass number of 649, revealing that the saccharide had a mass number of648.

[0110] The above non-reducing saccharide was hydrolyzed with sulfuricacid and then analyzed for sugar composition by conventional gaschromatography. As a result, only D-glucose was detected, revealing thatthe saccharide was cyclotetrasaccharide composed of four moles ofD-glucose in view of the above mass number and non-reducibility.

[0111] Nuclear magnetic resonance analysis (called “NMR”) of thenon-reducing saccharide gave a ¹H-NMR spectrum in FIG. 2 and a ¹³C-NMRspectrum in FIG. 3. These spectra were compared with those of knownsaccharides and revealed that they coincided with those ofcyclotetrasaccharide-as disclosed by Gregory L. Cote et al. in EuropeanJournal of Biochemistry, Vol. 226, pp. 641-648 (1994).

[0112] Based on these results, the non-reducing saccharide isolated inthe above method was identified with cyclotetrasaccharide, i.e.,cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}.

EXPERIMENT 2 α-isomaltosyl-Transferring Enzyme andα-Isomaltosylglucosaccharide-Forming Enzyme EXPERIMENT 2-1 EnzymePreparation Containing α-isomaltosyl-Transferring Enzyme andα-Isomaltosylglucosaccharide-Forming Enzyme

[0113] A liquid culture medium, consisting of 4.0% (w/v) of “PINE-DEX#4™”, a partial starch hydrolyzate commercialized by Matsutani ChemicalInd., Tokyo, Japan, 1.8% (w/v) of “ASAHIMEAST™”, a yeast extractcommercialized by Asahi Breweries, Ltd., Tokyo, Japan, 0.1% (w/v) ofdipotassium phosphate, 0.06% (w/v) of sodium dihydrogen phosphatedodecahydrate, 0.05% (w/v) magnesium sulfate heptahydrate, and water,was placed in 500-ml Erlenmeyer flasks in for 20 min, cooled, and thenseeded with a stock culture of Bacillus globisporus C9 strain, FERMBP-7143, followed by culturing under rotary-shaking conditions at 27° C.and 230 rpm for 48 hours for a seed culture.

[0114] About 20 L of a fresh preparation of the same liquid culturemedium as used in the above seed culture was placed in a 30-L fermenter,sterilized by heating, and then cooled to 27° C. and inoculated with 1%(v/v) of the seed culture, followed by culturing at 27° c. and a pH of6.0-8.0 for 48 hours under aeration-agitation conditions. The resultingculture was centrifuged at 10,000 rpm for 30 min to obtain about 18 L ofa supernatant. The obtained supernatant had an activity of about 1.5units/ml of α-isomaltosyl-transferring enzyme, an activity of about 0.45unit/ml of α-isomaltosylglucosaccharide-forming enzyme, and an activityof about 0.95 unit/ml of cyclotetrasaccharide-forming enzyme.

[0115] The supernatant was concentrated to give a volume of two litersby using “API-2013™”, a membrane for ultrafiltration commercialized byAsahi Kasei Corporation, Tokyo, Japan, to obtain an enzyme preparationcontaining α-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme. The enzyme preparation had about 8.5units/ml of cyclotetrasaccharide-forming activity.

EXPERIMENT 2-2 Purified α-Isomaltosyl-Transferring Enzyme

[0116] About 18 L of the culture supernatant of Bacillus globisporus C9strain in Experiment 2-1, having 26,900 units ofα-isomaltosyl-transferring enzyme, was salted out with 80% saturatedammonium sulfate at 4° C. for 24 hours. The formed sediments werecollected by centrifugation at 10,000 rpm for 30 min, dissolved in 10 mMphosphate buffer (pH 7.5), and dialyzed against a fresh preparation ofthe same buffer to collect a dialyzed inner solution.

[0117] The dialyzed inner solution was fed to a column packed with 1,000ml of “SEPABEADS FP-DA13™” gel, an ion-exchange resin commercialized byMitsubishi Chemical Industries, Ltd., Tokyo, Japan, followed bycollecting non-adsorbed fractions.

[0118] The non-adsorbed fractions were pooled and dialyzed against 10mM-phosphate buffer (pH 7.0) with 1 M ammonium sulfate, and the dialyzedinner solution was collected and centrifuged to remove insolublesubstances, and then fed to affinity column chromatography using 500 mlof “SEPHACRYL HR S-200™”, a gel commercialized by Amersham Corp., Div.Amersham International, Arlington Heights, Ill., USA, followed byfeeding thereunto a linear gradient decreasing from 1 M to 0 M ofammonium sulfate and collecting fractions, having anα-isomaltosyl-transferring enzyme activity, eluted at around 0 Mammonium sulfate.

[0119] The factions purified on the above affinity column chromatographywere pooled and dialyzed against 10 mM phosphate buffer (pH 7.0)containing 1 M ammonium sulfate. The dialyzed inner solution wascentrifuged to remove insoluble substances, and the resultingsupernatant was fed to hydrophobic chromatography using 350 ml of“BUTYL-TOYOPEARL 650 M”, a gel commercialized by Tosoh Corporation,Tokyo, Japan, followed by feeding thereunto a linear gradient decreasingfrom 1 M to 0 M of ammonium sulfate and collecting fractions, having anα-isomaltosyl-transferring enzyme activity, eluted at around 0.3 Mammonium sulfate.

[0120] The factions purified in the above hydrophobic chromatographywere pooled and dialyzed against 10 mM phosphate buffer (pH 7.0)containing 1 M ammonium sulfate. The dialyzed inner solution wascentrifuged to remove insoluble substances, and the resultingsupernatant was fed to affinity column chromatography again under thesame conditions as above, followed by collecting a fraction with theenzyme activity.

[0121] The faction, purified twice on the affinity columnchromatography, was subjected to SDS-PAGE using a 7.5% (w/v) ofpolyacrylamide gel, resulting in a single protein band at a positioncorresponding to a molecular weight of about 112,000±20,000 daltons. Thespecific activity of the enzyme in the fraction was calculated based onits enzyme activity and protein quantitative analysis, revealing that ithad a specific activity of about 26.9 units/mg protein. Thus, a purifiedα-isomaltosyl-transferring enzyme specimen was obtained.

EXPERIMENT 2-3 Purified α-Isomaltosylglucosaccharide-Forming Enzyme

[0122] About 18 L of a culture supernatant of Bacillus globisporus C9strain obtained by the method in Experiment 2-1, having 8,110 units ofan α-isomaltosylglucosaccharide-forming enzyme, was subjected tosalting-out with an 80% saturation solution of ammonium sulfate,dialyzed, and purified on ion-exchange resin according to method inExperiment 2-2.

[0123] The fractions purified on the ion-exchange resin were pooled anddialyzed against 10 mM phosphate buffer (pH 7.0) with 1 M ammoniumsulfate. The dialyzed inner solution was centrifuged to remove insolublesubstances, and fed to affinity column chromatography using 500 ml of“SEPHACRYL HR S-200”, a gel commercialized by Amersham Corp., Div.,Amersham International, Arlington Heights, Ill., USA. The elution of theenzyme was carried out using a linear gradient decreasing from 1 M to 0M of ammonium sulfate and subsequently a linear gradient increasing from0 mM to 100 mM of maltotetraose, followed by collecting fractions elutedat around 30 mM maltotetraose with anα-isomaltosylglucosaccharide-forming enzyme activity.

[0124] The fractions, purified on the above affinity columnchromatography, were pooled and dialyzed against 10 mM phosphate buffer(pH 7.0) containing 1 M ammonium sulfate. The dialyzed inner solutionwas centrifuged to remove insoluble substances, and the resultingsupernatant was fed to hydrophobic column chromatography using 350 ml of“BUTYL-TOYOPEARL 650 M”, a gel commercialized by Tosoh Corporation,Tokyo, Japan, followed by feeding thereunto a linear gradient decreasingfrom 1 M to 0 M of ammonium sulfate and collecting fractions with anα-isomaltosylglucosaccharide-forming enzyme activity eluted at around0.3 M ammonium sulfate.

[0125] The factions, purified on the above hydrophobic chromatography,were poled and dialyzed against 10 mM phosphate buffer (pH 7.0)containing 1 M ammonium sulfate. The dialyzed inner solution wascentrifuged to remove insoluble substances., and the resultingsupernatant was fed to affinity chromatography again under the sameconditions as in the above, followed by collecting a fraction with theenzyme activity.

[0126] The above faction was subjected to SDS-PAGE using a 7.5% (w/v) ofa polyacrylamide gel and detected as a single protein band with amolecular weight of about 140,000±20,000 daltons. The specific activityof the enzyme in the fraction was calculated based on its enzymeactivity and protein quantitative analysis, revealing that it had aspecific activity of about 13.6 units/mg protein. Thus, a purifiedspecimen of α-isomaltosylglucosaccharide-forming enzyme was obtained.

[0127] Though the above Experiments 2-1 to 2-3 show the data onpreparations of enzymes from Bacillus globisporus C9 strain, suchenzymes can be also obtained from Bacillus globisporus C₁₋₁ strain, FERMBP-7144, in accordance with the above methods. The following are theproperties of enzymes purified from Bacillus globisporus C11 strain inaccordance with Experiments 2-1 to 2-3:

[0128] (A) α-isomaltosyl-transferring enzyme Molecular weight onSDS-PAGE: 102,000±20,000 Daltons Specific activity: About 26.9 units/mgprotein

[0129] (B) α-isomaltosylglucosaccharide-forming enzyme Molecular weighton SDS-PAGE: 137,000±20,000 Daltons Specific activity: About 13.4units/mg protein

EXPERIMENT 3 Production of Cyclotetrasaccharide by Enzymatic Action onPartial Starch Hydrolyzate and Branched Cyclotetrasaccharide as aBy-Product EXPERIMENT 3-1 Enzymatic Reaction

[0130] 3.7 kg of “PINE-DEX #100™”, a partial starch hydrolyzatecommercialized by Matsutani Chemical Ind., Tokyo, Japan, was dissolvedin 35 L of 10 mM sodium acetate (pH 6.0). To the resulting solution wasadded about 17,500 units of cyclotetrasaccharide forming activity of anenzyme preparation, obtained by the method in Experiment 2-1, andenzymatically reacted at 30° C. for two days, followed by boiling for 20min to inactivate the remaining enzyme. The resulting mixture was cooledto 45° C. and admixed with 11 g (137,500 units) of “NEO-SPITASE PK2™”,an α-amylase specimen commercialized by Nagase Biochemicals, Ltd.,Kyoto, Japan, and 44 g (140,800 units) of “GLUCOZYME™”, a glucoamylasespecimen commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, andthen adjusted to pH 6.0, followed by enzymatically reacting at 45° C.for one day. The reaction mixture was boiled for 20 minutes toinactivate the remaining enzymes, and then cooled and filtered in ausual manner to obtain a filtrate. The filtrate was concentrated to givea solid concentration of about 16% (w/w) by using a reverse osmosismembrane. The resulting concentrate was subjected in a usual manner todecoloration, desalting, filtration, and concentration to obtain about6.1 kg of a saccharide solution with a solid content of about 3.5 kg.

[0131] The saccharide solution was analyzed by the following HPLC byusing both cyclotetrasaccharide, isolated by the method in Experiment 1,and known saccharides as a standard. HPLC was run using “CCPM”, achromatograph commercialized by Tosoh Corporation, Tokyo, Japan, “AQ-303ODS”, and a column with a diameter of 4.6 mm and a length of 25 cmcommercialized by YMC Co., Ltd., Tokyo, Japan, at a column temperatureof 40 and a flow rate of 0.5 ml/min of water; and using “RI-8012™”, adifferential refractometer commercialized by Tosoh Corporation, Tokyo,Japan. Among the ingredients detected on this analysis, the data forthose with a respectively-wide peak area were shown in Table 2 includingtheir retention times, names, and relative values of peak areas. TABLE 2Retention time on HPLC (min*) Name Peak area** 10.5 Cyclotetrasaccharide43.8% 19.7 Secondary Product 1 10.6% 24.7 Secondary Product 2 3.3% 49.9Secondary Product 3 0.5% 58.2 Secondary Product 4 0.3%

[0132] Upon the above HPLC, as a whole, there exists a tendency that thehigher the molecular weight of a compound tested, the longer theretention time of the compound becomes. Thus, the results in Table 2show that the enzymatic reaction of this experiment usually formssaccharides with a higher molecular weight than cyclotetrasaccharide,i.e., those which are composed of a larger number of saccharides thancyclotetrasaccharide, together with cyclotetrasaccharide.

EXPERIMENT 3-2 Preparation of Cyclotetrasaccharides

[0133] 6.1 kg of a saccharide solution obtained by the method inExperiment 3-1 was fed to columns, consisting of ten columns, which eachhad a diameter of 13.5 cm and a length of 160 cm and which were cascadedin series and packed with about 225 L of “AMBERLITE CR-1310 (Na-form)™”,an ion-exchange resin commercialized by Japan Organo Co., Ltd., Tokyo,Japan; and chromatographed at a flow rate of about 45 L/h of water and acolumn temperature of 60° C. The resulting elute was fractionated andanalyzed for saccharide composition on HPLC as described in Experiment3-1 in such a manner of pooling fractions relatively rich incyclotetrasaccharide into a saccharide solution with a solid content of1,530 g. HPLC analysis of the saccharide solution conducted under thesame conditions as above and the data calculated based on the results onthe peak areas of HPLC revealed that the pooled fraction, i.e., a highcyclotetrasaccharide content fraction, contained 79.8%cyclotetrasaccharide of 79.8% and 6.1% isomaltose to the total sugarcontents.

[0134] The high cyclotetrasaccharide content fraction with a solidcontent of 1,310 g was adjusted to pH 5.0 and 50° C., and then incubatedfor 20 hours after admixed with 1,000 units/g solids of“TRANSGLUCOSIDASE L AMANO™”, an α-glucosidase commercialized by AmanoPharmaceutical Co., Ltd., Aichi, Japan, and 60 units/g solids of“GLUCOTEAM™”, a glucoamylase commercialized by Nagase Biochemicals,Ltd., Kyoto, Japan. After removing insoluble substances by filtration,the above reaction mixture was desalted with “DIAION PK218™”, a cationexchange resin commercialized by Mitsubishi Chemical Industries, Ltd.,Tokyo, Japan, and “AMBERLITE IRA411™”, an anion exchange resincommercialized by Japan Organo Co., Ltd., Tokyo, Japan, followed byconcentrating. The concentrate was fractionated according to theconditions used in the above chromatography to obtain a fraction with acyclotetrasaccharide purity of at least 97%. The fractions were pooledand in a conventional manner decolored, desalted, filtered, andconcentrated into a saccharide solution with a solid content of about1,260 g. After adjusted to give a solid concentration of about 50%(w/w), the saccharide solution was placed in a cylindrical plasticvessel and cooled from 65° C. to 20° C. over about 20 hours under gentlestirring conditions to effect crystallization. The formed crystals werecollected by separation and dried at 60° C. for three hours under normalpressure to obtain 544 g of a crystalline powder. The crystalline powderwas of a crystalline cyclotetrasaccharide with a purity of 99.9% and amoisture content of 12.7%.

EXPERIMENT 3-3 Isolation of By-Products

[0135] 6.1 kg of a saccharide solution obtained by the method inExperiment 3-1 was fractionated according to the chromatography asdescribed in Experiment 3-2, and the resulting fractions were analyzedfor saccharide composition on HPLC as described in Experiment 3-1.Fractions relatively rich in the by-products 1 and 2 were pooled forFraction 1, while fractions relatively rich in the by-products 3 and 4were pooled for Fraction 2. Fractions 1 and 2 had solid contents of 320g and 150 g, respectively. Based on the peak areas of Fractions 1 and 2on HPLC, their saccharide compositions were determined, revealing thatFraction 1 contained 47.9% of the by-product 1 and 14.9% of theby-product 2 to the total sugars and Fraction 2 contained components,which contained the by-products 3 and 4 had a retention time longer thanthat of by-product 2 when measured on the above HPLC, in an amount of atleast 25% to the total sugars.

[0136] Fraction 1 was kept at a pH of 11 or higher by the addition ofsodium hydroxide and heated at 95° C. or higher for one hour todecompose reducing saccharides, followed by decoloring, desalting,filtering, and concentrating in a conventional manner. An adequateamount of the concentrate equal to a solid content of 50 g by weight wassubjected to preparative liquid chromatography using “YMN-PACK ODS-AR355-15 S-15 120A”, a preparative column commercialized by YMC Co.,Ltd., Tokyo, Japan, and a purified, deionized water as a moving phase.Based on the HPLC analysis described in Experiment 3-1, the abovechromatography yielded a fraction with a solid content of 20 gcontaining the by-product 1 with a purity of at least 97%, and anotherfraction with a solid content of five grams containing the by-product 2with a purity of at least 96%.

[0137] Similarly as in Fraction 1, the above Fraction 2 was subjected todecomposition of reducing saccharides, followed by decoloring,desalting, filtrating, and concentrating. An adequate amount of theconcentrate equal to a solid content of 10 g by weight was subjected topreparative liquid chromatography by using preparative chromatographysimilarly as in Fraction 1. Based on the HPLC analysis described inExperiment 3-1, the above chromatography yielded a fraction with a solidcontent of 77 mg containing the by-product 3 with a purity of at least97%, and another fraction with a solid content of 77 mg containing theby-product 4 with a purity of at least 97%.

EXPERIMENT 3-4 Identification of By-Products

[0138] Fractions rich in the by-products 1 to 4 obtained in Experiment3-3 were subjected to wholly or partly to the following analysis: (1)Mass number was determined by mass spectroscopy using the high-speedatom shocking method, (2) reducing power was determined by theSomogyi-Nelson method, (3) saccharide composition was determined byconventional gas chromatography to analyze constituent saccharides afterhydrolysis with sulfuric acid, (4) biological analysis was conducted bythe HPLC analysis described in Experiment 3-1 after treatment with‘TRANSGLUCOSIDASE L AMANO™’, an α-glucosidase commercialized by AmanoPharmaceutical Co., Ltd., Aichi, Japan, (5) binding fashion wasdetermined by a conventional methylation analysis, (6) specific rotationwas measured in a conventional manner, and (7) ¹³C-NMR was done in aconventional manner. The results are as follows:

[0139] The by-product 1 was a substantially non-reducing saccharide witha mass number of 810. Considering the mass number and the fact that theconstituent saccharides were only D-glucose molecules, the by-product 1was estimated to be composed of five D-glucose molecules. When treatedwith α-glucosidase, cyclotetrasaccharide and an equimolar of glucosewere formed. Upon methylation analysis, 2,3-dimethylated compound,2,3,4-trimethylated compound, 2,4,6-trimethylated compound, and2,3,4,6-tetramethylated compound were detected in a molar ratio of0.83:1.02:1.69:1, meaning a composition ratio of 1:1:2:1. The by-product1 had a specific rotation of [α]²⁵d+246° and a ¹³C-NMR spectrum in FIG.4. The data on the signal assignment of the by-product 1 is in Table 4below together with those of cyclotetrasaccharide and other saccharidesin Experiments 3-4 and 4-3.

[0140] Based on these results, the by-product 1 was identified with ablanched cyclotetrasaccharide having a structure represented by ChemicalFormula 1.

[0141] The by-product 2 was a substantially non-reducing saccharide witha mass number of 972. Considering the mass number and the fact that theconstituent saccharides were D-glucose molecules, the by-product 2-wasestimated to be composed of six D-glucose molecules. Upon methylationanalysis, 2,4-dimethylated compound, 0.2,3,4-trimethylated compound,2,4,6-trimethylated compound, and 2,3,4,6-tetramethylated compound weredetected in a molar ratio of 0.94:2.01:1.72:1, meaning a compositionratio of 1:2:2:1. By-product 2 had a specific rotation of [α]²⁵d+246°and a ¹³C-NMR spectrum in FIG. 5. The data on the signal assignment ofthe by-product 2 is in Table 4 below together with those ofcyclotetrasaccharide and other saccharides in Experiments 3-4 and 4-3.

[0142] Based on these results, the by-product 2 was identified with ablanched cyclotetrasaccharide having a structure represented by ChemicalFormula 3.

[0143] The by product 3 was a substantially non-reducing saccharide witha mass number of 1,296. Considering the mass number and the fact thatthe constituent saccharides were D-glucose molecules, the by-product 3was estimated to be composed of eight D-glucose molecules and had a¹³C-NMR spectrum in FIG. 6. The data on the signal assignment of theby-product 3 is in Table 4 below together with those ofcyclotetrasaccharide and other saccharides in Experiments 3-4 and 4-3.

[0144] Based on these results, the by-product 3 was identified with ablanched cyclotetrasaccharide having a structure represented by ChemicalFormula 4.

[0145] The by-product 4 was a substantially non-reducing saccharide witha mass number of 1, 134. Considering the mass number and the fact thatthe constituent saccharides were D-glucose molecules, the by-product 4was estimated to be composed of seven D-glucose molecules. Uponmethylation analysis, 2,3-dimethylated compound, 2,4-dimethylatedcompound, 2,3,4-trimethylated compound, 2,4,6-trimethylated compound,and 2,3,4,6-tetramethylated compound were detected in molar ratio of0.78:0.78:1.47:1.60:2, meaning a composition ratio of 1:1:1:2:2. Theby-product 4 gave a ¹³C-NMR spectrum in FIG. 7. The data on the signalassignment of the by-product 3 is in Table 4 below together with thoseof cyclotetrasaccharide and other saccharides in Experiments 3-4 and4-3.

[0146] Based on these results, the by-product 4 was identified with ablanched cyclotetrasaccharide having a structure represented by ChemicalFormula 5.

[0147] Judging totally the constituent saccharides and the bindingfashions of blanched parts of blanched cyclotetrasaccharides identifiedin Experiment 3-4, the blanched cyclotetrasaccharides as by-products,which were formed in the enzymatic reaction in Experiment 3-1, wouldhave the following characteristics. The blanched cyclotetrasaccharideshave a basic structure represented by Formula 1, where one or more of R₁to R₁₂ are optionally substitutedα-D-glucopyranosyl-(1→6)-{α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-}_(n)α-D-glucopyranosyl groups, with the proviso that “n” is an integer of atleast 0 and, when at least two of the groups of R₁ to R₁₂ are the abovegroups, the number of each “n” is independent in each group; and inrelatively many cases, R₂ and/or R₈ are/is optionally substitutedα-D-glucopyranosyl-(1→6)-{α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-}_(n)α-D-glucopyranosyl groups, with the proviso that “n” is an integer of atleast 0 and, when both R₂ and R₈ are the above groups, the number ofeach “n” is independent in each group.

EXPERIMENT 4 Glycosyl Transfer to Cyclotetrasaccharide by IsolatedEnzyme EXPERIMENT 4-1 Glycosyl Transfer byα-Isomaltosylglucosaccharide-Forming Enzyme

[0148] A 10 mM sodium acetate buffer (pH6.0) containing 20% (w/w) ofcyclotetrasaccharide, obtained by the method in Experiment 3-2, and 10%(w/w) of maltopentaose, produced by Hayashibara Biochemical LaboratoriesInc., Okayama, Japan, was incubated at 30° C. for 24 hours after admixedwith 3 units/g maltopentaose of a purifiedα-isomaltosylglucosaccharide-forming enzyme obtained by the method inExperiment 2-3. The reaction mixture was then boiled for 20 minutes toinactivate the remaining enzyme.

[0149] The resulting mixture was adjusted to pH 5.0 and incubated at 50°C. for one hour after admixed with 500 units/g solids of “GLUCOTEAM™”, aglucoamylase commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan.Thereafter, the reaction mixture was boiled for 10 minutes to inactivatethe remaining enzyme.

[0150] After glucoamylase treatment, the resulting reaction mixture wasfiltered in a conventional manner with a membrane, desalted, andanalyzed on HPLC as in Experiment 3-1. Comparison of retention times onHPLC for the blanched cyclotetrasaccharides, which had been isolated andidentified in Experiments 3-3 and 3-4, revealed that the reactionmixture contained a branched cyclotetrasaccharide, represented byChemical Formula 1, in an amount of 17.3% to the total sugars, whencalculated based on a relative ratio of its peak area on HPLC. Thisresult indicates that the branched cyclotetrasaccharide of the presentinvention can be efficiently produced by contactingα-isomaltosylglucosaccharide-forming enzyme with cyclotetrasaccharide.

EXPERIMENT 4-2 Glycosyl Transfer by α-Isomaltosyl-Transferring Enzyme

[0151] A 10 mM sodium acetate buffer (pH6.0) containing 20% (w/w)cyclotetrasaccharide obtained by the method in Experiment 3-2 and 10%(w/w) of panose produced by Hayashibara Biochemical Laboratories Inc.,Okayama, Japan, was incubated at 30° C. for 24 hours after admixed with30 units/g panose of a purified α-isomaltosyl-transferring enzymeobtained by the method in Experiment 2-2. The reaction mixture wasboiled for 20 minutes to inactivate the remaining enzyme.

[0152] The reaction mixture was in a conventional manner filtered with amembrane, desalted, and analyzed on HPLC as shown in Experiment 3-1.Comparison of retention times on HPLC of the blanchedcyclotetrasaccharides, which had been isolated and identified inExperiments 3-3 and 3-4, revealed that the reaction mixture contained abranched cyclotetrasaccharide, represented by Chemical Formula 3, in anamount of 4.9% to the total sugars, when calculated based on a relativeratio of its peak area on HPLC. This result revealed that the branchedcyclotetrasaccharide of the present invention can be efficientlyproduced by contacting α-isomaltosyl-transferring enzyme withcyclotetrasaccharide.

EXPERIMENT 4-3 Glycosyl Transfer by Cyclomaltodextrin EXPERIMENT 4-3(a)Enzymatic Reaction

[0153] Ten grams of cyclotetrasaccharide obtained by the method inExperiment 3-2 and 10 g of α-cyclodextrin, produced by HayashibaraBiochemical Laboratories Inc., Okayama, Japan, were dissolved in 30 g of50 mM sodium acetate buffer (pH 5.5), and then incubated at 50° C. for24 hours after admixed with 10 units/g α-cyclodextrin of CGTase fromBacillus stearothermophilus, commercialized by Hayashibara BiochemicalLaboratories Inc., Okayama, Japan. The reaction mixture was boiled for20 minutes to inactivate the remaining enzyme.

[0154] To the reaction mixture was added 350 g of 50 mM sodium acetatebuffer (pH 4.5) and then incubated at 40° C. for four hours afteradmixed with 2,000 units of “GLUCOTEAM™”, a glucoamylase commercializedby Nagase Biochemicals, Ltd., Kyoto, Japan. Thereafter, the resultingculture was boiled for 20 minutes to inactivate the remaining enzyme.

[0155] The above reaction mixtures, obtained after the CGTase treatmentand the combination treatment with CGTase and glucoamylase, weresubjected to HPLC as in Experiment 3-1. While, under the same conditionsas above, cyclotetrasaccharide and glucose were analyzed. Based onthese, the components in the above reaction mixtures were identified.The results on the reaction mixtures after the CGTase treatment and thecombination treatment with CGTase and glucoamylase are respectivelyshown in FIG. 8a and FIG. 8b.

[0156] A peak X with a retention time of about 10 min, commonly observedin FIGS. 8a and 8 b, is a peak for cyclotetrasaccharide, and a peak Gwith a retention time of about six minutes, specifically observed inFIG. 8b, is a peak for glucose. As shown in FIGS. 8a and 8 b, thereaction mixture with the CGTase treatment contained newly formedcomponents with longer retention times than that of cyclotetrasaccharide(FIG. 8a), while that with the combination treatment of CGTase andglucoamylase has almost lost these components except for two componentswith peaks 1 and 2 (FIG. 8b). These results indicate that the peaks,observed in FIG. 8a, except for that for cyclotetrasaccharide were forglycosyl derivatives of cyclotetrasaccharide to which one- or -moreglycosyl groups were bound. In FIG. 8b, the two peaks, i.e., peaks 1 and2, with retention times longer than that of cyclotetrasaccharide areglucosyl derivatives of cyclotetrasaccharide where a glycosyl group isbound to a specific position of cyclotetrasaccharide. The retentiontimes of these two components in the reaction mixture, received with thecombination treatment of CGTase and glucoamylase, are shown in Table 3together with respective names and relative values of peak areas. TABLE3 Retention time on HPLC (min*) Name Peak area** 18.7 CGTase product 1About 35% 38.7 CGTase product 2 About 22%

EXPERIMENT 4-3(b) Isolation and Identification of Reaction Product

[0157] A reaction mixture, obtained after the combination treatment ofCGTase and glucoamylase in the method in Experiment 4-3(a), was filteredwith a membrane, and the filtrate was desalted with “DIAION PK218™”, acation exchange resin commercialized by Mitsubishi Chemical Industries,Ltd., Tokyo, Japan; and “AMBERLITE IRA411™”, an anion exchange resincommercialized by Japan Organo Co., Ltd., Tokyo, Japan, followed byconcentrating. The concentrate was fractionated according to thechromatographic conditions in Experiment 3-2, and the resulting eachfraction was analyzed on HPLC in Experiment 3-1 to obtain a fractioncontaining product 1 or 2 produced by CGTase (called “CGTase product 1or 2”) with a purity of 97%.

[0158] The fraction rich in the CGTase product 1 was in a conventionalmanner subjected to ¹³C-NMR, and the obtained spectrum coincided withthat of the by-product 1 of Chemical Formula 1 (FIG. 4) in Experiment3-4. Based on the result, the CGTase product 1 was identified with abranched cyclotetrasaccharide represented by Chemical formula 1.

[0159] The fraction rich in the CGTase product 2 was analyzed inaccordance with the 7th analytical item in Experiment 3-4. The CGTaseproduct 2 was a substantially non-reducing saccharide with a mass numberof 972. Considering the mass number and the fact that the constituentsaccharides were D-glucose molecules, the CGTase product 2 was judged tobe composed of six molecules of D-glucose. When treated withα-glucosidase, the CGTase product 2 was hydrolyzed intocyclotetrasaccharide and glucose in a molecule ratio of 1:2. Uponmethylation analysis, 2,3-dimethylated compound, 2,4,6-trimethylatedcompound, and 2,3,4,6-tetramethylated compound were observed in a molarratio of 0.89:1:1.24, meaning a composition ratio [α]²⁵d+241° and gave a¹³C-NMR spectrum in FIG. 9. The assignment of signals of the spectrumare in Table 4 below together with the those of cyclotetrasaccharide andother saccharides described in Experiments 3-4. TABLE 4 Chemical shift(ppm) Carbon number A B C D E F 1a 99.34 99.24 99.46 99.40 99.44 99.492a 74.28 75.49 72.84 72.87 72.80 75.72 3a 75.45 75.75 82.47 82.98 82.4575.98 4a 73.35 81.18 73.81 73.72 73.76 81.38 5a 72.78 71.14 72.45 72.4672.38 71.34 6a 70.22 70.27 70.17 70.10 69.98 70.47 1b 101.20 101.18101.17 101.08 101.13 101.43 2b 72.64 72.71 72.64 72.63 72.58 72.91 3b77.31 77.58 77.23 77.13 77.26 77.93 4b 73.62 73.58 73.62 73.65 73.5273.76 5b 74.23 74.22 74.25 74.28 74.20 74.42 6b 62.88 62.87 62.88 62.8762.84 63.08 1c — 99.35 99.32 99.28 99.18 — 2c — 74.22 74.25 74.28 75.47— 3c — 75.49 75.45 75.43 75.77 — 4c — 73.34 73.36 73.37 81.14 — 5c —72.71 72.78 72.75 71.09 — 6c — 70.15 70.08 70.00 70.20 — 1d — 101.18101.17 101.08 101.13 — 2d — 72.65 72.64 72.63 72.66 — 3d — 77.37 77.2377.04 77.51 — 4d — 73.58 73.57 73.58 73.52 — 5d — 74.22 74.25 74.2874.20 — 6d — 62.87 62.88 62.87 62.84 — 1e — 102.14 100.59 100.50 100.54102.34 2e — 74.40 74.25 74.28 74.20 74.60 3e — 75.62 75.82 75.85 75.7775.81 4e — 72.23 72.16 72.02 72.12 72.43 5e — 74.10 73.10 73.08 73.0574.33 6e — 63.38 68.18 67.85 68.14 63.58 1f — — 101.84 102.17 101.79 —2f — — 74.25 72.63 74.20 — 3f — — 75.82 82.98 75.77 — 4f — — 72.25 72.8772.20 — 5f — — 74.50 74.28 74.45 — 6f — — 63.18 62.87 63.13 — 1g — — —100.50 102.11 — 2g — — — 74.28 74.36 — 3g — — — 75.85 75.57 — 4g — — —72.24 72.20 — 5g — — — 72.99 74.06 — 6g — — — 67.85 63.33 — 1h — — —102.17 — — 2h — — — 74.28 — — 3h — — — 75.92 — — 4h — — — 72.24 — — 5h —— — 74.51 — — 6h — — — 63.16 — —

[0160] *: By-products 1 to 4 mean those which were isolated inExperiment 3-3, and CGTase 2 means the CGTase product 2 which wasisolated in Experiment 4-3(b).

[0161] Based on these results, the CGTase product 2 was identified witha branched cyclotetrasaccharide having a structure represented byChemical formula 2.

[0162] Totally judging from the constituent saccharides and the bindingfashions of the branched part of the branched cyclotetrasaccharideidentified in Experiment 4-3(b) and from the results of HPLC analysis onthe branched cyclotetrasaccharide after the enzymatic reaction inExperiment 4-3(a), it is considered that the branchedcyclotetrasaccharide formed by the enymatic reaction in Experiment4-3(a) has the following characteristics: The blanchedcyclotetrasaccharide has a basic structure represented by Formula 1,wherein in Formula 1 one or more of R₁ to R₁₂, particularly, R₁ and/orR₇ in relatively many cases are/is an optionally substituted{α-D-glucopyranosyl-(1→4)-}_(n) α-D-glucopyranosyl group(s), with theproviso that “n” is an integer of 0 or more, and when at least two of R₁to R₁₂ are the above groups, “n” is each independent in each group.

EXPERIMENT 4-4 Glycosyl Transfer by β-Galactosidase from Bacilluscirculans EXPERIMENT 4-4(a) Enzymatic Reaction

[0163] Twenty grams of cyclotetrasaccharide obtained by the method inExperiment 3-2 and 20 g of a special grade lactose, commercialized in93.3 g of 20 mM sodium acetate buffer (pH 6.0). The solution wassubjected to an enzymatic reaction at 40° C. for 24 hours after admixedwith 3 units/g lactose of “BIOLACTAN 5™”, a β-galactosidase from amicroorganism of the species Bacillus circulans commercialized by DaiwaKasei K.K., Osaka, Japan, followed by boiling the resulting mixture for20 minutes to inactivate the remaining enzyme.

[0164] The reaction mixture thus obtained and an aqueous solution ofcyclotetrasaccharide were analyzed on HPLC in Experiment 3-1. As aresult, the HPLC analysis revealed the formation of at least three kindsof new components, which had different retention times from that ofcyclotetrasaccharide (about 10 minutes), in the reaction mixture. Table5 shows the retention times of the three components together with theirnames and relative values of peak areas. TABLE 5 Retention time on HPLC(min*) Name Peak area** 14.0 β-Galactosidase product 1 12.2% 19.7β-Galactosidase product 2 2.9% 20.3 β-Galactosidase product 3 1.1%

EXPERIMENT 4-4(b) Isolation and Identification of Reaction Product

[0165] To a reaction mixture obtained by the method in Experiment 4-4(a)was added 4.8 g of sodium hydroxide and kept at 100° C. for one hour todecompose reducing saccharides. The resulting mixture was decolored,desalted, filtered, and concentrated in a conventional manner, followedby subjecting the concentrate to a preparative liquid chromatographyusing “YMN-PACK ODS-AQR355-15AQ, S-10/20 μm, 120A™”, a preparativecolumn commercialized by YMC Co., Ltd., Tokyo, Japan, and using apurified, deionized water as a moving bed. The eluate was analyzed onHPLC as described in Experiment 3-1, resulting in obtaining a fractioncontaining a product produced by β-galactosidase (or β-galactosidaseproduct 1) with a purity of at least 97%.

[0166] The above fraction was analyzed in accordance with sevenanalytical items in Experiment 3-4. The β-galactosidase product 1 was asubstantially non-reducing saccharide with a mass number of 810. Theconstituent saccharides of the β-galactosidase product 1 were D-glucoseand D-galactose which were present in a composition ratio of 4:1.Judging from its mass number, the β-galactosidase product 1 wasconsidered to be composed of four molecules of D-glucose and onemolecule of D-galactose. The β-galactosidase product 1 had a specificrotation of [α]²⁵d+200° and a ¹³C-NMR signals of the spectrum togetherwith those of cyclotetrasaccharide and other results in Experiments 4-5to 4-7.

[0167] Based on these results, the β-galactosidase product 1 wasidentified with a branched cyclotetrasaccharide represented by ChemicalFormula 6.

EXPERIMENT 4-5(a) Enzymatic Reaction

[0168] Twenty grams of cyclotetrasaccharide obtained by the method inExperiment 3-2 and 20 g of a special grade lactose, commercialized byWako Pure Chemical Industries, Ltd., Tokyo, Japan, were dissolved in93.3 g of 20 mM sodium acetate buffer (pH 4.5), and then incubated at40° C. for 24 hours after admixed with 10 units/g lactose of “LACTASEYA-O™”, a β-galactosidase from a microorganism of the speciesAspergillus niger, commercialized by Yakult Pharmaceutical Inc. Co.,Ltd., Tokyo, Japan. Thereafter, the resulting reaction mixture wasboiled for 20 min to inactivate the remaining enzyme.

[0169] The reaction mixture thus obtained and an aqueous solution ofcyclotetrasaccharide were analyzed on HPLC described in Experiment 3-1.As a result, the HPLC analysis revealed the formation of at least threekinds of new components, which had different retention times from thatof cyclotetrasaccharide (about 10 minutes), in the reaction mixture.Table 7 shows the retention times of the three components together withtheir names and relative values of peak areas. TABLE 6 Retention time onHPLC (min*) Name Peak area** 14.1 Chemical Formula 6*** 3.3% 15.1β-Galactosidase product 4 0.7% 19.1 β-Galactosidase product 5 7.1%

EXPERIMENT 4-5(b) Isolation and Identification of Reaction Product

[0170] To the reaction mixture, obtained by the method in Experiment4-5(a), was added 4.8 g of sodium hydroxide and kept at 100° C. for onehour to decompose reducing saccharides. The resulting mixture wasdecolored, desalted, filtered, and concentrated in a conventionalmanner. The concentrate was subjected to preparative liquidchromatography using “YMN-PACK ODS-AQR355-15AQ, S-10/20 μm, 120A”, apreparative column commercialized by YMC Co., Ltd., Tokyo, Japan, andusing a purified, deionized water as a moving bed. The eluate wasanalyzed on HPLC described in Experiment 3-1, resulting in obtaining afraction containing a product formed by β-galactosidase (orβ-galactosidase product 4) with a purity of at least 94%, and anotherfraction containing a product formed by β-galactosidase (orβ-galactosidase product 5) with a purity of at least 99%.

[0171] The above fraction of β-galactosidase product 4 was analyzed inaccordance with the seven analytical items in Experiment 3-4. Theβ-galactosidase product 4 was a substantially non-reducing saccharidewith a mass number of 973. The constituent saccharides were D-glucoseand D-galactose, which were present in a molar ratio of 2:1. Judgingfrom its mass number, the β-galactosidase product 4 was considered to becomposed of four molecules of D-glucose and two molecules ofD-galactose. Upon methylation analysis, 2,4-dimethylated glucose,2,3,4-trimethylated glucose, 2,4,6-trimethylated glucose,2,3,4-trimethylated galactose, and 2,3,4,6-tetramethylated galactosewere detected in a molar ratio of 1:1.86:0.96:1.34:1.12, meaning acomposition ratio of 1:2:1:1:1. Upon ¹³C-NMR, the β-galactosidaseproduct 4 gave a spectrum of FIG. 11. Table 7 shows the assignment ofsignals of the spectrum together with those of cyclotetrasaccharide andother results in Experiments 4-5 to 4-7.

[0172] Based on these results, the β-galactosidase product 4 wasidentified with a branched cyclotetrasaccharide represented by ChemicalFormula 8.

[0173] The above fraction rich in the β-galactosidase product 5 wasanalyzed in accordance with the seven analytical items in Experiment3-4. The β-galactosidase product 5 was a substantially non-reducingsaccharide with a mass number of 810. The constituent saccharides wereD-glucose and D-galactose present in a composition ratio of 4:1. Judgingfrom its mass number, the β-galactosidase product 4 was considered to becomposed of four molecules of D-glucose and one molecule of D-galactose.Upon methylation analysis, 2,4-dimethylated glucose, 2,3,4-trimethylatedglucose, 2,4,6-trimethylated glucose, and 2,3,4,6-tetramethylatedgalactose were detected in a molar ratio of 1:2.02:1.00:1.04, meaning acomposition ratio of 1:2:1:1. Upon ¹³C-NMR, the β-galactosidase product5 gave a spectrum of FIG. 12. Table 7 shows the assignment of signals ofthe spectrum together with those of cyclotetrasaccharide and otherresults in Experiments 4-5 to 4-7.

[0174] Based on these results, the β-galactosidase product 5 wasidentified with a branched cyclotetrasaccharide represented by ChemicalFormula 7.

[0175] Judging totally from the constituent saccharide is and thebinding fashion of the branched part of the branchedcyclotetrasaccharide identified in Experiment 4-5(b), the branchedcyclotetrasaccharide formed by the enzymatic reaction in Experiment4-5(a) has the following characteristics in general. The blanchedcyclotetrasaccharide has a basic structure represented by Formula 1,wherein in Formula 1 one or more of R₁ to R₁₂, particularly in manycases, R₆ and/or R₁₂ are/is an optionally substituted{β-D-galactopyranosyl-(1→6)-}_(n) α-D-galactopyranosyl group(s), withthe proviso that “n” is an integer of at least 0 and it is independentwhen at least two of R₁ to R₁₂ have the above groups.

EXPERIMENT 4-6 Glycosyl Transfer by α-Galactosidase EXPERIMENT 4-6(a)Enzymatic Reaction

[0176] Five grams of cyclotetrasaccharide obtained by the method inExperiment 3-2 and five grams of a special grade melibiose,commercialized by Wako Pure Chemical Industries, Ltd., Tokyo, Japan,were dissolved in 15 g of 50 mM sodium acetate buffer (pH 5.0), and thenincubated at 40° C. for 30 hours after admixed with 100 units/gmelibiose of “MELIBIASE™”, an α-galactosidase from a microorganism ofthe genus Mortierella, commercialized by Hokkaido Sugar Co., Ltd.,Tokyo, Japan, and the reaction mixture was boiled for 20 min toinactivate the remaining enzyme.

[0177] The reaction mixture and an aqueous solution ofcyclotetrasaccharide were subjected to HPLC described in Experiment 3-1.As a result, the HPLC analysis revealed the formation of at least onenewly formed component with a retention time of 13.3 min, which differedfrom cyclotetrasaccharide with a retention time of about 10 min. Uponthe HPLC analysis, the newly formed component had a peak area of about1.0% to the total peak areas.

EXPERIMENT 4-6(b) Isolation and Identification of Reactive Product

[0178] After a reaction mixture obtained by the method in Experiment4-6(a) was in a conventional manner desalted, filtered, andconcentrated, the resulting concentrate was subjected to preparativeliquid chromatography described in Experiment 4-5(b) and to HPLCanalysis described in Experiment 3-1, followed by collecting a fractioncontaining a product, formed with α-galactosidase (or α-galactosidaseproduct), with a purity of at least 98%.

[0179] The above fraction rich in α-galactosidase product was analyzedaccording to seven analytical items in Experiment 3-4. Theα-galactosidase product was a substantially non-reducing saccharide witha mass number of 810. The α-galactosidase product had constituentsaccharides of D-glucose and D-galactose in a composition ratio of 4:1.Judging from its mass number, the α-galactosidase product was consideredto be composed of four molecules of D-glucose and one molecule ofD-galactose. Upon methylation analysis, 2,4-dimethylated glucose,2,3,4-trimethylated glucose, 2,4,6-trimethylated glucose, and2,3,4,6-tetramethylated galactose were observed in a molar ratio of1:2.09:1.02:1.02, meaning a composition ratio of 1:2:1:1. Theα-galactosidase product gave a ¹³C-NMR spectrum in FIG. 13. Table 7shows the assignment of signals of the spectrum together with those ofcyclotetrasaccharide and other results in Experiments 4-5 to 4-7.

[0180] Based on these results, the α-galactosidase product wasidentified with a branched cyclotetrasaccharide represented by ChemicalFormula 9.

EXPERIMENT 4-7 Glycosyl Transfer by Lysozyme EXPERIMENT 4-7(a) EnzymaticReaction

[0181] Twenty grams of cyclotetrasaccharide obtained by the method inExperiment 3-2 and 10 g of “NA-COS-Y™”, a chichin oligosaccharidecommercialized by Yaizu Suisankagaku Industry Co., Ltd., Shizuoka,Japan, containing about 55%, d.s.b., of a chitin oligosaccharide with apolymerization degree of two to six, were dissolved in 45 g of 50 mMsodium acetate buffer (pH 4.5), and then incubated at 60° C. for ninedays after admixed with 2.8 g of albumin lysozyme, commercialized bySeikagaku Corporation, Tokyo, Japan. Thereafter, the reaction mixturewas boiled for 20 min to inactivate the remaining enzyme.

[0182] As a treatment before HPLC, the resulting reaction mixture wascentrifuged, then the supernatant was filtered by using “SEP-0013™”, anultrafiltration membrane commercialized by Asahi Kasei Corporation,Tokyo, Japan, to remove proteins and desalted in a conventional manner.The pretreated solution and an aqueous solution of cyclotetrasaccharidewere subjected to HPLC described in Experiment 3-1. As a result, theHPLC analysis revealed the formation of at least one newly formedcomponent with a retention time of 36.6 min, which differed fromcyclotetrasaccharide with a retention time of about 10 min. Upon theHPLC analysis, the newly-formed component had a peak area of about 7.3%to the total peak areas.

EXPERIMENT 4-7(b) Isolation and Identification of Reactive Product

[0183] A reaction mixture, obtained by the method in Experiment 4-7(a),was similarly treated as the pretreatment of HPLC in Experiment 4-7(a),and then subjected to preparative liquid chromatography according to themethod in Experiment 4-5(b), and to HPLC analysis described inExperiment 3-1, followed by collecting a fraction containing a productformed by lysozyme (or a lysozyme product), with a purity of at least99%, after analysis and confirmation on the HPLC analysis in Experiment3-1.

[0184] The above fraction rich in the lysozyme product was analyzedaccording to the seven analytical items in Experiment 3-4. The lysozymeproduct was a substantially non-reducing saccharide with a mass numberof 851. The lysozyme product had D-glucose and N-acetyl-D-glucosamine(N-acetyl-D-chitosamine) as constituent saccharides in a compositionratio of 4:1. Judging from its mass number, the lysozyme product wasconsidered to be composed of four molecules of D-glucose and onemolecule of N-acetyl-D-glucosamine. Upon methylation analysis,2,4-dimethylated glucose, 2,3,4-trimethylated glucose, and2,4,6-trimethylated glucose were observed in a molar ratio of1.02:1:1.67, meaning a composition ratio of 1:1:2. The product had aspecific rotation of [α]²⁵d+246° and a ¹³C-NMR spectrum of FIG. 14.Table 7 shows the assignment of signals of the product together withthat of cyclotetrasaccharide and other results described in Experiments4-4 to 4-6. TABLE 7 Chemical shift (ppm) Carbon number A β-Gal1* β-Gal4*β-Gal5* α-Gal* Lys* 1a 99.34 98.96 99.39 99.36 99.25 99.47 2a 74.2873.67 74.27 74.24 74.28 72.48 3a 75.45 83.59 75.45 75.42 75.44 84.53 4a73.35 73.11 73.28 73.24 73.42 74.03 5a 72.78 72.44 72.76 72.76 72.6072.57 6a 70.22 70.06 70.38 70.35 70.10 70.18 1b 101.20 101.04 101.22101.27 101.07 101.13 2b 72.64 72.60 72.65 72.62 72.60 72.69 3b 77.3177.29 77.37 77.36 77.35 77.22 4b 73.62 73.62 73.34 73.31 73.61 73.65 5b74.23 74.21 73.46 73.45 72.79 74.23 6b 62.88 62.86 70.66 70.59 67.9462.84 1c — 99.21 99.39 99.36 99.25 99.31 2c — 74.26 74.27 74.24 74.2874.28 3c — 75.41 75.45 75.42 75.44 75.44 4c — 73.34 73.22 73.24 73.3573.35 5c — 72.70 72.76 72.73 72.60 72.77 6c — 70.06 70.22 70.20 70.1070.18 1d — 100.95 101.22 101.21 100.96 101.13 2d — 72.54 72.57 72.5472.60 72.63 3d — 77.03 77.24 77.21 77.18 77.22 4d — 73.53 73.34 73.3173.61 73.60 5d — 74.21 74.27 74.24 74.22 74.23 6d — 62.86 62.88 62.8662.87 62.84 1e — 107.57 105.93 106.01 100.55 — 2e — 74.08 73.42 73.5871.20 — 3e — 75.18 75.26 75.42 72.29 — 4e — 71.46 71.37 71.34 71.97 — 5e— 78.02 76.57 77.83 73.75 — 6e — 64.18 71.60 63.70 63.87 — 1f — — 106.02— — 104.66 2f — — 73.62 — — 58.43 3f — — 75.45 — — 76.38 4f — — 71.31 —— 71.86 5f — — 77.87 — — 78.52 6f — — 63.67 — — 63.38 CO — — — — —177.58 CH₃ — — — — — 24.92

[0185] and 5 isolated in Experiment 4-5(b), α-Gal means α-galactosidaseproduct isolated in Experiment 4-6(b), and Lys means lysozyme productisolated in Experiment 4-7(b).

[0186] Based on these results, the lysozyme product obtained in thisExperiment was identified with a branched cyclotetrasacchariderepresented by Chemical Formula 10.

EXPERIMENT 5 Crystal of Branched Cyclotetrasaccharide

[0187] Fraction rich in any one of the by-product 1, obtained by themethod in Experiment 3-3 (a branched cyclotetrasaccharide represented byChemical Formula 1, and hereinafter, the branched cyclotetrasaccharidesof the present invention are respectively shown by their ChemicalFormula numbers.), the by-product 2 (Chemical Formula 3), the CGTaseproduct 2 obtained by the method in Experiment 4-3(b) (Chemical Formula2), the β-galactosidase product 1 obtained by the method in Experiment4-4(b) (Chemical Formula 6), and the β-galactosidase product 5 obtainedby the method in Experiment 4-5(b) (Chemical Formula 7), wasconcentrated, resulting in an observation of crystal. After collectingeach crystal, they were dried at ambient temperature into five kinds ofcrystals of the above branched cyclotetrasaccharides. The HPLC analysisdescribed in Experiment 3-1 revealed that the crystals of ChemicalFormulae 1, 2, 6, and 7 had purities of at least 99%, and the one ofChemical Formula 3 had a purity of at least 98%.

[0188] The crystalline powders were respectively analyzed on thefollowing whole or part of the items of crystalline form, X-raydiffraction, color, moisture content, melting point, and thermalproperty. The crystalline form was microscopically observed. The X-rayanalysis was examined by a conventional powdery X-ray diffractionanalysis. The moisture content was measured by the Karl Fischer method,and the melting point was measured by “MODEL MP-21™”, a melting pointmeasurement device commercialized by Yamato Scientific Co., Ltd., Tokyo,Japan. The thermal property was analyzed based on thermogravimetricanalysis using “TG/DTA220 type™”, a digital thermoanalyzercommercialized by Seiko Instruments Inc., Chiba, Japan.

[0189] The microscopic observation revealed that the compounds ofChemical Formulae 1, 2, and 3 had a pillar-, needle-, and pillar-form,respectively.

[0190] The data on X-ray diffraction analysis of all the above crystalsare in FIGS. 15 to 19. Predominant diffraction angles (2θ) observed inthe analysis are tabulated in Table 8 along with the results on color,moisture content, and melting point. TABLE 8 Number* of Main diffractionMoisture crystal of Compound angles (2θ) Color content water Meltingpoint Chemical Formula 1  8.1°, 12.2° White 11.1% 5 to 6 Not measurable,as it was decomposed 14.2°, 15.4° at around 270° C. Chemical Formula 2 5.6°, 8.8° White 17.5% 11 to 12 Not measurable, as it was decomposed16.9°, 21.9° at around 280° C. Chemical Formula 3  7.9°, 12.1° White15.8% 10 to 11 Not measurable, as it was decomposed 17.9°, 20.2° ataround 275° C. Chemical Formula 6 11.0°, 12.3° White 17.1%  9 to 10 93°C. 12.8°, 24.9° Chemical Formula 7  8.7°, 13.0° White 11.0% 5 to 6 Notmeasurable, as it was decomposed 21.7°, 26.1° at around 245° C.

[0191] The results of thermal properties of all the above crystals arerespectively shown in FIG. 20 to 24. As shown in FIG. 20, the compoundof Chemical Formula 1 gave a weight reduction corresponding to that offour moles of water as the temperature increased to 150° C., and gave aweight reduction due to its decomposition at a temperature of around300° C. or over. These results indicate that the compound of ChemicalFormula 1 in the form of a penta- or hexa-hydrous crystal is convertedinto a mono- or di-hydrous crystal when heated to 150° C. under normalpressure.

[0192] As shown in FIG. 21, the compound of Chemical Formula 2 gave aweight reduction corresponding to that of six to seven moles of water asthe temperature increased to about 150° C., and also gave a weightreduction due to its decomposition at a temperature of around 300° C. orover. These results indicate that the compound of Chemical Formula 2 inthe form of a undeca- or dodeca-hydrous crystal is converted into atetra- or penta-hydrous crystal when heated to 150° C. under normalpressure.

[0193] As shown in FIG. 22, the compound of Chemical Formula 3 gave aweight reduction corresponding to that of six to seven moles of water asthe temperature increased to about 110° C., and also gave a weightreduction due to its decomposition at a temperature of around 300° C. orover. These results indicated that the compound of Chemical Formula 3 inthe form of a deca- or undeca-hydrous crystal is converted into a tri-or tetra-hydrous crystal when heated to 110° C. under normal pressure.

[0194] As shown in FIG. 23, the compound of Chemical Formula 6 gave aweight reduction corresponding to that of seven to eight moles of wateras the temperature increased to about 120° C., and also gave a weightreduction due to its decomposition at a temperature of around 300° C. orover. These results indicate that the compound of Chemical Formula 6 inthe form of a nona- or deca-hydrous crystal is converted into a mono- ordi-hydrous crystal when heated to 120° C. under normal pressure.

[0195] As shown in FIG. 24, the compound of Chemical Formula 7 gave aweight reduction corresponding to that of five to six moles of water asthe temperature increased to about 130° C., and also gave a weightreduction due to its decomposition at a temperature of around 300° C. orover. These results indicate that the compound of Chemical Formula 7 inthe form of a penta- or hexa-hydrous crystal is converted into ananhydrous or monohydrous crystal when heated to 130° C. under normalpressure.

EXAMPLE A-1 Syrup Containing Branched Cyclotetrasaccharide Representedby Chemical Formulae 1 and 2

[0196] One part by weight of cyclotetrasaccharide obtained by the methodin Experiment 3-2 and one part by weight of α-cyclodextrin,commercialized by Hayashibara Biochemical Laboratories Inc., Okayama,Japan, were dissolved in three parts by weight of 50 mM sodium acetatebuffer (pH 5.5). The resulting solution was incubated at 50° C. for 24hours after admixed with 10 units/g α-cyclodextrin of CGTase from amicroorganism of the species Bacillus stearothermophilus, commercializedby Hayashibara Biochemical Laboratories Inc., Okayama, Japan, and thereaction mixture was boiled for 20 minutes to inactivate the remainingenzyme.

[0197] To the reaction mixture was added 350 g of 50 mM sodium acetatebuffer (pH 4.5) and then incubated at 40° C. for four hours afteradmixed with 200 units of “GLUCOTEAM™”, a glucoamylase specimencommercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, per gram ofthe initially added α-cyclodextrin. Thereafter, the resulting mixturewas boiled for 20 min to inactivate the remaining enzyme.

[0198] The reaction mixture thus obtained was membrane filtered,desalted with “DIAION PK218™”, a cation exchange resin commercialized byMitsubishi Chemical Industries, Ltd., Tokyo, Japan, and “AMBERLITEIRA411™”, an anion exchange resin commercialized by Japan Organo Co.,Ltd., Tokyo, Japan, and concentrated. The concentrate was fractionatedby chromatography according to the conditions used in Experiment 3-2,and the resulting each fraction was analyzed on the HPLC in Experiment3-1 to collect a fraction containing a branched cyclotetrasaccharide,represented by Chemical Formulae 1 and 2, with a purity of 85% or highereach. The fraction was concentrated into a syrup with a solidconcentration of about 50% (w/w).

[0199] The syrup can be advantageously used as a material for productsin a variety fields of foods, beverages, cosmetics, and pharmaceuticalswithout any treatment or after concentrated, dried, crystallized, orpulverized into a product in the form of an amorphous powder, molasses,block, etc.

EXAMPLE A-2 Syrup Containing Branched Cyclotetrasaccharide Representedby Chemical Formula 3

[0200] Two parts by weight of cyclotetrasaccharide obtained by themethod in Experiment 3-2 and one part by weight of panose commercializedby Hayashibara Biochemical Laboratories Inc., Okayama, Japan, weredissolved in seven parts by weight of 10 mM sodium acetate buffer (pH6.0). The resulting solution was incubated at 30° C. for 24 hours afteradmixed with 30 units/g panose of a purified α-isomaltosyl-transferringenzyme obtained by the method in Experiment 2-2, and the reactionmixture was boiled for 20 min to inactivate the remaining enzyme.

[0201] The reaction mixture thus obtained was membrane filtered,desalted with “DIAION PK218™”, a cation exchange resin commercialized byMitsubishi Chemical Industries, Ltd., Tokyo, Japan, and “AMBERLITEIRA411™”, an anion exchange resin commercialized by Japan Organo Co.,Ltd., Tokyo, Japan, and concentrated. The concentrate was fractionatedby chromatography according to the conditions in Experiment 3-2, and theresulting each fraction was analyzed on the HPLC in Experiment 3-1 tocollect a fraction with a branched cyclotetrasaccharide, represented byChemical Formula 3, with a purity of 80% or higher. The fraction wasconcentrated into a syrup with a solid concentration of about 40% (w/w).

[0202] The syrup can be advantageously used as a material for productsin a variety fields of foods, beverages, cosmetics, and pharmaceuticalswithout any treatment or after concentrated, dried, crystallized, orpulverized into a product in the form of an amorphous powder, molasses,block, etc.

EXAMPLE A-3 Syrup Containing Branched Cyclotetrasaccharide Representedby Chemical Formula 6

[0203] Two parts by weight of cyclotetrasaccharide obtained by themethod in Experiment 3-2 and two parts by weight of lactose were mixedand dissolved in nine parts by weight of 20 mM sodium acetate buffer (pH6.0). The resulting solution was incubated at 40° C. for 24 hours afteradmixed with three units/g lactose of “BIOLACTAN™”, a β-galactosidasespecimen from a microorganism of the species Bacillus circulanscommercialized by Daiwa Kasei K. K., Osaka, Japan, and the reactionmixture was boiled for 20 minutes to inactivate the remaining enzyme.

[0204] To the reaction mixture thus obtained was-added 0.5 part byweight of sodium hydroxide and kept at 100° C. for one hour to decomposereducing saccharides. The obtained reaction mixture was in aconventional manner desalted, filtered, and concentrated. Theconcentrate was subjected to preparative liquid chromatography using“YMC-PACK ODS-AQR355-15AQ S-10/20 μm, 120A™”, a preparative columncommercialized by YMC Co., Ltd., Tokyo, Japan, where a purified,deionized water was used as a moving phase. The eluate was analyzed onthe HPLC described in Experiment 3-1 to collect a fraction containing abranched cyclotetrasaccharide, represented by Chemical Formula 6, with apurity of at least 85%. The fraction was concentrated to obtain a syrupwith a solid concentration of 40% (w/w).

[0205] The syrup can be advantageously used as a material for productsin variety fields of foods, beverages, cosmetics, and pharmaceuticalswithout any treatment or after concentrated, dried, crystallized, orpulverized into a product in the form of an amorphous powder, molasses,block, etc.

EXAMPLE A-4 Syrup Containing Branched Cyclotetrasaccharide Representedby Chemical Formula 7

[0206] Two parts by weight of cyclotetrasaccharide by the method inExperiment 3-2 and two parts by weight of lactose were dissolved in nineparts by weight of 20 mM sodium acetate buffer (pH 4.5). The resultingsolution was incubated at 40° C. for 24 hours after admixed with 10units/g lactose of “LACTASE YA-O™”, a β-galactosidase specimen from amicroorganism of the species Aspergillus niger commercialized by YakultPharmaceutical Inc. Co., Ltd., Tokyo, Japan, and the reaction mixturewas boiled for 20 min to inactivate the remaining enzyme.

[0207] To the reaction mixture thus obtained was added 0.5 part byweight of sodium hydroxide and kept at 100° C. for one hour to decomposereducing saccharides. The resulting reaction mixture was in aconventional manner desalted, filtrated, and concentrated. Theconcentrate was subjected to preparative liquid chromatography using“YMC-PACK ODS-AQR355-15AQ, S-10/20 μm 120A™”, a preparative columncommercialized by YMC Co., Ltd., Tokyo, Japan, where a purified,deionized water was used as a moving phase. The eluate was analyzed onthe HPLC described in Experiment 3-1 to collect a fraction containing abranched cyclotetrasaccharide, represented by Chemical Formula 7, with apurity of at least 85%. The fraction was concentrated into a syrup witha solid concentration of 45% (w/w).

[0208] The syrup can be advantageously used as a material for productsin variety fields of foods, beverages, cosmetics, and pharmaceuticalswithout any treatment or after concentrated, dried, crystallized, orpulverized into a product in the form of an amorphous powder, molasses,block, etc.

EXAMPLE A-5 Crystal of Branched Cyclotetrasaccharide Represented byChemical Formula 1 or 2

[0209] According to Example A-1, a reaction mixture, obtained throughthe sequential treatments of CGTase and glucoamylase and theinactivation of enzyme, was subjected to chromatographic separationsimilarly as in Example A-1. The eluate was analyzed on the HPLC inExperiment 3-1 to collect fractions containing branchedcyclotetrasaccharides, represented by Chemical Formulae 1 and 2, with apurity of 97% or higher each. The fractions were respectivelyconcentrated and then admixed with a corresponding crystal of ChemicalFormula 1 or 2 obtained in Experiment 5 as a seed crystal to effectsufficient crystallization. The crystals were centrifuged and collectedin a conventional manner, and the collected crystals were dried atambient temperature to obtain respective crystals of branchedcyclotetrasaccharides, represented by Chemical Formulae 1 and 2, with apurity of at least 99% each. Measurement of moisture content describedin Experiment 5 revealed that the compound of Chemical Formula 1 was apenta- or hexa-hydrous crystal, while the compound of Chemical Formula 2was an undeca- or dodeca-hydrous crystal.

[0210] The crystals can be arbitrarily used as materials for products invariety fields of foods, beverages, cosmetics, and pharmaceuticals.

EXAMPLE A-6 Crystal of Branched Cyclotetrasaccharide Represented byChemical Formula 3

[0211] According to Example A-2, a reaction mixture, obtained throughthe treatments of β-galactosidase and the inactivation of enzyme, wassubjected to chromatographic separation similarly as in Example A-2. Theeluate was analyzed on the HPLC described in Experiment 3-1 to collect afraction containing branched cyclotetrasaccharides, represented byChemical Formula 3, with a purity of at least 97%. The fraction wasconcentrated and then admixed with the crystal of the compound ofChemical Formula 3 obtained in Experiment 5 as a seed crystal to effectsufficient crystallization. The formed crystal was centrifuged andcollected in a conventional manner, and the collected crystal was driedat ambient temperature to obtain a crystal of a branchedcyclotetrasaccharide, represented by Chemical Formula 3, with a purityof at least 99%. Measurement of moisture content described in Experiment5 revealed that the crystal was a deca- or undeca-hydrous crystal.

[0212] The crystal can be arbitrarily used as a material for products invariety fields of foods, beverages, cosmetics, and pharmaceuticals.

EXAMPLE A-7 Crystal of Branched Cyclotetrasaccharide Represented byChemical Formula 6

[0213] According to Example A-3, a reaction mixture, obtained throughthe treatments of α-isomaltosyl-transferring enzyme and the inactivationof enzyme, was subjected to chromatographic separation similarly as inExample A-3. The eluate was analyzed on the HPLC described in Experiment3-1 to collect a fraction containing branched cyclotetrasaccharides,represented by Chemical Formula 6, with a purity of at least 96%. Thefraction was concentrated and then admixed with the crystal of thecompound of Chemical Formula 6 obtained in Experiment 5 as a seedcrystal to effect sufficient crystallization. The crystal wascentrifuged and collected in a conventional manner, and the collectedcrystal was dried at ambient temperature to obtain a crystal of abranched cyclotetrasaccharide, represented by Chemical Formula 6, with apurity of at least 99%. Measurement of moisture content described inExperiment 5 revealed that the crystal was a nona- or deca-hydrouscrystal.

[0214] The crystal can be arbitrarily used as a material for products invariety fields of foods, beverages, cosmetics, and pharmaceuticals.

EXAMPLE A-8 Crystal of Branched Cyclotetrasaccharide Represented byChemical Formula 7

[0215] According to Example A-4, a reaction mixture, obtained throughthe treatments of β-galactosidase and the inactivation of enzyme, wassubjected to chromatographic separation similarly as in Example A-4. Theeluate was analyzed on the HPLC described in Experiment 3-1 to collect afraction containing branched cyclotetrasaccharides, represented byChemical Formula 7, with a purity of at least 97%. The fraction wasconcentrated and admixed with the crystal of the compound of ChemicalFormula 7 obtained in Experiment 5 as a seed crystal to effectsufficient crystallization. The crystal was centrifuged and collected ina conventional manner, and the collected crystal was dried at ambienttemperature to obtain a crystal of a branched cyclotetrasaccharide,represented by Chemical Formula 7, with a purity of at least 99%.Measurement of moisture described in Experiment 5 revealed that thecrystal was a penta- or hexa-hydrous crystal.

[0216] The crystal can be arbitrarily used as a material for products invariety fields of foods, beverages, cosmetics, and pharmaceuticals.

EXAMPLE A-9 Syrup Containing Branched Cyclotetrasaccharides

[0217] 3.7 kg of “PINE-DEX #100™”, a partial starch hydrolyzatecommercialized by Matsutani Chemical Ind., Tokyo, Japan, was dissolvedin 35 L of 10 mM sodium acetate buffer (pH 6.0), and the solution wasincubated at 30° C. for two days after admixed with 17,500 units of acyclotetrasaccharide-forming activity of an enzyme preparation obtainedby the method in Experiment 2-1, and the reaction mixture was boiled for20 minutes to inactivate the remaining enzyme. The reaction mixture wascooled to 45° C. and admixed with 11 g (137,500 units) of“NEO-SPITASE™”, an α-amylase specimen commercialized by NagaseBiochemicals, Ltd., Kyoto, Japan, and 44 grams (140,800 units) of“GLUCOZYME™”, a glucoamylase specimen commercialized by NagaseBiochemicals, Ltd., Kyoto, Japan, and then adjusted to pH 6.0, followedby enzymatically reacting at 45° C. for one day. The reaction mixturethus obtained was boiled for 20 minutes to inactivate remaining enzymes,and then cooled and filtered in a conventional manner to obtain afiltrate. The filtrate was concentrated to give a solid concentration ofabout 16% (w/w) by using a reverse osmosis membrane. The concentrate wassubjected in a conventional manner to decoloration, desalting,filtration, and concentration to obtain about 6.1 kg of a syrupcontaining 3.5 kg of solids consisting of 12% of branchedcyclotetrasaccharides, 44% of cyclotetrasaccharide, 25% of glucose, and19% of oligosaccharides.

[0218] Since the syrup is substantially free of crystallization andeasily produced on an industrial scale and at a lesser cost, it can bearbitrarily used as a material for products in a variety fields offoods, beverages, cosmetics, and pharmaceuticals.

EXAMPLE A-10 Syrup Containing Branched Cyclotetrasaccharides

[0219] 6.1 kg of a saccharide solution, obtained by the method inExperiment 3-1, was fed to ten columns, having an inner diameter of 13.5cm and a length of 160 cm each, which had been packed with about 225 Lof “AMBERLITE CR-1310 (Na-form)™”, an ion-exchange resin commercializedby Japan Organo Co., Ltd., Tokyo, Japan, and cascaded in series, andchromatographed at a flow rate of about 45 L/h of water and at a columntemperature of 60° C. The eluate from the columns was fractionated anddetermined for saccharide composition by the HPLC described inExperiment 3-1. Fractions, which were relatively rich incyclotetrasaccharide, were collected and pooled to obtain a saccharidesolution with a solid content of about 1,530 g. The saccharide solution,i.e., a high cyclotetrasaccharide content fraction, was analyzed on theHPLC under the same conditions as above, and based on the peak areasdetermined on the HPLC analysis, it was composed of 79.8% ofcyclotetrasaccharide and. 6.1% of isomaltose to the total sugars.

[0220] The saccharide solution in an amount equal to a solid content of1,310 g was adjusted to pH 5.0 and 50° C., and then incubated for 20hours after admixed with 1,000 units/g solids of “TRANSGLUCOSIDASE LAMANO™”, an α-glucosidase specimen commercialized by AmanoPharmaceutical Co., Ltd., Aichi, Japan, and 60 units/g solids of‘GLUCOZYME™’, a glucoamylase specimen commercialized by NagaseBiochemicals, Ltd., Kyoto, Japan. After removing insoluble substances byfiltration, the above enzymatic reaction mixture was desalted with“DIAION PK218™”, a cation exchange resin commercialized by MitsubishiChemical Industries, Ltd., Tokyo, Japan, and “AMBERLITE IRA411™”, ananion exchange resin commercialized by Japan Organo Co., Ltd., Tokyo,Japan, and then concentrated. The concentrate was fractionated accordingto the conditions as used in the above chromatographic separation tocollect a fraction of cyclotetrasaccharide with a purity of 97%. Thefraction was in a conventional manner decolored, desalted, filtered, andconcentrated to obtain a saccharide solution with a solid content of1,260 g. After adjusted to a solid concentration of about 50% (w/w), thesaccharide solution was placed in a cylindrical plastic vessel andcooled from 65° C. to 20° C. over about 20 hours under gentle stirringconditions. The resulting mixture was subjected to separation, and theobtained mother liquor was purified and concentrated into a syrup with asolid concentration of about 45%, consisting of 4% of branchedcyclotetrasaccharides, 94% of cyclotetrasaccharide, 1% of glucose, and1% of other saccharides.

[0221] Since the syrup is substantially free of crystallization andeasily produced on an industrial scale and at a lesser cost, it can bearbitrarily used as a material for products in a variety fields offoods, beverages, cosmetics, and pharmaceuticals.

EXAMPLE A-11 Syrup Containing Branched Cyclotetrasaccharides

[0222] To 400 g of a syrup containing branched cyclotetrasaccharidesobtained in Example A-9 was added 0.1 g/g solids of “N154™”commercialized by Nikki Chemical Co., Ltd., Kanagawa, Japan, anactivated Raney nickel catalyst with an alkali. The mixture was placedin an autoclave and hydrogenated by reacting at 100° C. for four hoursand further at 120° C. for two hours while stirring and keeping at ahydrogen pressure of 100 kg/cm². After cooled, the resultinghydrogenated mixture was taken out from the autoclave and filtered bypassing through an activated carbon layer with about one centimeter inthickness to remove the Raney nickel catalyst. The filtrate was in aconventional manner decolored with an activated charcoal, desalted withion-exchange resins in H- and OH-forms, purified, and concentrated togive a concentration of about 40% and to obtain a syrup which wassubstantially free of crystallization and composed of 12% of branchedcyclotetrasaccharides, 44% of cyclotetrasaccharide, 25% of sorbitol, and19% of other saccharides.

[0223] Since the syrup is substantially free of crystallization andeasily produced on an industrial scale and at a lesser cost, it can bearbitrarily used as a material for products in a variety fields offoods, beverages, cosmetics, and pharmaceuticals.

EXAMPLE A-12 Syrup of Branched Cyclotetrasaccharides

[0224] A substantially non-reducing and non-crystallizing syrup, whichhad a solid concentration of about 55% and consisted of 4% of branchedcyclotetrasaccharides, 94% of cyclotetrasaccharide, 1% of sorbitol, and1% of other saccharides, was obtained similarly as in Example A-11except for replacing 400 g of the syrup containing branchedcyclotetrasaccharides obtained in Example A-9 with 400 g of the syrupcontaining branched cyclotetrasaccharides obtained in Example A-10.

[0225] Since the syrup is substantially free of crystallization andeasily produced on an industrial scale and at a lesser cost, it can bearbitrarily used as a material for products in a variety fields offoods, beverages, cosmetics, and pharmaceuticals.

EXAMPLE B-1 Sweetener

[0226] To 0.8 part by weight of a branched cyclotetrasaccharide crystal,penta- or hexa-hydrate, represented by Chemical Formula 1, obtained bythe method in Example A-5, were homogeneously added 0.2 part by weightof “TREHA™”, a crystalline trehalose hydrate commercialized byHayashibara Shoji Inc., Okayama, Japan, 0.01 part by weight of “a GSWEET™”, an α-glycosylstevioside product commercialized by Toyo SugarRefining Co., Tokyo, Japan, and 0.01 part by weight of “ASPALTAME™”, aproduct of L-aspartyl-L-phenylalanine methyl ester, followed by feedingthe resulting mixture to a granulator to obtain a granular sweetener.The product has a satisfactory sweetness and an about two-fold highersweetening power of sucrose. Since the branched cyclotetrasaccharide ishardly digestible and ferfmentable and is substantially free fromcalorie the product has only about {fraction (1/10)} calorie of that ofsucrose with respect to sweetening power. In addition, the product issubstantially free of deterioration and stable even when stored at roomtemperature. Thus, the product is preferable as a high quality, lowcaloric, less cariogenic sweetener.

EXAMPLE B-2 Hard Candy

[0227] One hundred parts by weight of a 55% (w/w) sucrose solution weremixed while heating with 50 parts by weight of a syrup containing abranched cyclotetrasaccharide represented by Chemical Formula 6,obtained by the method in Example A-3. The mixture was then concentratedby heating under a reduced pressure to give a moisture content of lessthan 2%. The concentrate was mixed with 0.6 part by weight of citricacid and-an adequate amount of a lemon flavor, followed by forming theresultant into the desired product in a conventional manner. The productis a stable, high quality hard candy which has a satisfactory mouthfeel, taste, and flavor; less adsorbs moisture; and does neither inducecrystallization of sucrose nor cause melting.

EXAMPLE B-3 Beverage with Lactic Acid Bacteria

[0228] Fifty parts by weight of a syrup containing a branchedcyclotetrasaccharide represented by Chemical Formula 7, obtained by themethod in Example A-4, and 175 parts by weight of a skim milk powder,and 50 parts by weight of “NYUKAOLIGO™”, a high lactosucrose contentpowder commercialized by Hayashibara Shoji Inc., Okayama, Japan, weredissolved in 1,150 parts by weight of water. The resulting solution wassterilized at 65° C. for 30 min, then cooled to 40° C., inoculated in ausual manner with 30 parts by weight of lactic acid bacteria as astarter, and incubated at 37° C. for eight hours to obtain a beveragewith lactic acid bacteria. The product can be suitably used as a lacticacid beverage which has a satisfactory flavor and taste, containsoligosaccharides and cyclotetrasaccharide, stably retains the lacticacid bacteria, and has actions of promoting the growth of the bacteriaand controlling the intestinal conditions.

EXAMPLE B-4 Toothpaste

[0229] A syrup containing a branched cyclotetrasaccharide represented byChemical Formula 1, obtained by the method in Example A-1, was adjustedto a solid concentration of about 30% (w/w). A toothpaste was preparedby mixing 13 parts by weight of water with 15 parts by weight of theabove syrup, 45 parts by weight of calcium secondary phosphate, 1.5parts by weight of sodium lauryl sulfate, 25 parts by weight ofglycerol, 0.5 part by weight of polyoxyethylene sorbitan laurate, 0.02part by weight of saccharine, 0.05 part by weight of an antiseptic, and13 parts by weight of water. The product has an improved after taste anda satisfactory feeling after use without reducing the detergent power ofthe surfactant.

EXAMPLE B-5 Bath Salt

[0230] One part by weight of a peel juice of “yuzu” (a Chinese lemon)was admixed with 10 parts by weight of a branched cyclotetrasaccharidecrystal, undeca- or dodeca-hydrate, represented by Chemical Formula 2,obtained by the method in Example A-5, followed by drying andpulverizing the mixture into a powder containing a yuzu peel extract. Abath salt was prepared by mixing five parts by weight of the abovepowder with 90 parts by weight of grilled salt, two parts by weight ofhydrous crystalline trehalose, one part by weight of silicic anhydride,and 0.5 part by weight of “αG HESPERIDIN”, an α-glucosyl hesperidinproduct commercialized by Hayashibara Shoji, Inc., Okayama, Japan. Theproduct is a bath salt with an elegant, gentle flavor and a superiorskin moisturizing effect.

EXAMPLE B-6 Cosmetic Cream

[0231] Two parts by weight of polyoxyethylene glycol monostearate, fiveparts by weight of glyceryl monostearate, self-emulsifying, two parts byweight of a branched cyclotetrasaccharide crystal, deca- orundeca-hydrate, represented by Chemical Formula 3, obtained by themethod in Example A-6, one part by weight of “αG RUTIN”, an α-glucosylrutin product commercialized by Hayashibara Shoji, Inc., Okayama, Japan,one part by weight of liquid petrolatum, 10 parts by weight of glyceryltri-2-ethylhexanoate, and an adequate amount of an antiseptic weredissolved by heating in a usual manner. The resulting solution wasadmixed with two parts by weight of L-lactic acid, five parts by weightof 1,3-butylene glycol, and 66 parts by weight of refined water. Theresultant mixture was emulsified by a homogenizer and admixed with anadequate amount of a flavor while stirring to obtain a cosmetic cream.The product exhibits an antioxidant activity and has a relatively highstability, and these render it advantageously useful as a high qualitysunscreen, skin-refining agent, and skin-whitening agent.

EXAMPLE B-7 Tablet

[0232] Fourteen parts by weight of a branched cyclotetrasaccharidecrystal, nona- or deca-hydrate, represented by Chemical Formula 6,obtained by the method in Example A-7, were sufficiently mixed with 50parts by weight of aspirin and four parts by weight of corn starch. Themixture was then in a conventional manner tabletted by a tabulatingmachine into a tablet, 680 mg, 5.25 mm in thickness each. The tablet,processed with the filler-imparting ability of the branchedcyclotetrasaccharide, has a quite low hygroscopicity, sufficientphysical strength, and superior degradability in water.

INDUSTRIAL APPLICABILITY

[0233] As described above, the present invention was made based oncompletely novel findings by the present inventors that glycosylderivatives of cyclotetrasaccharide are formed as by-products ofcyclotetrasaccharide when the novel enzymes, i.e.,α-isomaltosyl-transferring enzyme andα-isomaltosylglucosaccharide-forming enzyme, which the present inventorshad previously found, are allowed to act on starch hydrolyzates; andthat a variety of glycosyl derivatives are obtained by allowingsaccharide-related enzymes such as the above-identified enzymes,cyclomaltodextrin glucanotransferase, β-galactosidase, α-galactosidase,and lysozyme to act on cyclotetrasaccharide Since the glycosylderivatives provided by the present invention, i.e., branchedcyclotetrasaccharides, have the intrinsic properties ofcyclotetrasaccharide such as an inclusion ability and substantiallynon-digestibility, they can be advantageously used in a various fieldsof foods and beverages, cosmetics, and pharmaceuticals similarly ascyclotetrasaccharide. Advanced analysis of physical and chemicalproperties and functions of the branched cyclotetrasaccharides of thepresent invention will give an important finding that leads todevelopment of novel uses of cyclotetrasaccharide and improvement of theproperties and functions thereof.

[0234] The present invention with these outstanding functions andeffects is a significant and important invention that greatlycontributes to this art.

1 2 1 3282 DNA Bacillus globisporus CDS (1)..(3282) sig_peptide(1)..(87) 1 atg tat gta agg aat cta aca ggt tca ttc cga ttt tct ctc tctttt 48 Met Tyr Val Arg Asn Leu Thr Gly Ser Phe Arg Phe Ser Leu Ser Phe 15 10 15 ttg ctc tgt ttc tgt ctc ttc gtc ccc tct att tat gcc att gat ggt96 Leu Leu Cys Phe Cys Leu Phe Val Pro Ser Ile Tyr Ala Ile Asp Gly 20 2530 gtt tat cat gcg cca tac gga atc gat gat ctg tac gag att cag gcg 144Val Tyr His Ala Pro Tyr Gly Ile Asp Asp Leu Tyr Glu Ile Gln Ala 35 40 45acg gag cgg agt cca aga gat ccc gtt gca ggc gat act gtg tat atc 192 ThrGlu Arg Ser Pro Arg Asp Pro Val Ala Gly Asp Thr Val Tyr Ile 50 55 60 aagata aca acg tgg ccc att gaa tca gga caa acg gct tgg gtg acc 240 Lys IleThr Thr Trp Pro Ile Glu Ser Gly Gln Thr Ala Trp Val Thr 65 70 75 80 tggacg aaa aac ggt gtc aat caa gct gct gtc gga gca gca ttc aaa 288 Trp ThrLys Asn Gly Val Asn Gln Ala Ala Val Gly Ala Ala Phe Lys 85 90 95 tac aacagc ggc aac aac act tac tgg gaa gcg aac ctt ggc act ttt 336 Tyr Asn SerGly Asn Asn Thr Tyr Trp Glu Ala Asn Leu Gly Thr Phe 100 105 110 gca aaaggg gac gtg atc agt tat acc gtt cat ggc aac aag gat ggc 384 Ala Lys GlyAsp Val Ile Ser Tyr Thr Val His Gly Asn Lys Asp Gly 115 120 125 gcg aatgag aag gtt atc ggt cct ttt act ttt acc gta acg gga tgg 432 Ala Asn GluLys Val Ile Gly Pro Phe Thr Phe Thr Val Thr Gly Trp 130 135 140 gaa tccgtt agc agt atc agc tct att acg gat aat acg aac cgt gtt 480 Glu Ser ValSer Ser Ile Ser Ser Ile Thr Asp Asn Thr Asn Arg Val 145 150 155 160 gtgctg aat gcg gtg ccg aat aca ggc aca ttg aag cca aag atc aac 528 Val LeuAsn Ala Val Pro Asn Thr Gly Thr Leu Lys Pro Lys Ile Asn 165 170 175 ctttcc ttt acg gcg gat gat gtc ctc cgc gta cag gtt tct cca acc 576 Leu SerPhe Thr Ala Asp Asp Val Leu Arg Val Gln Val Ser Pro Thr 180 185 190 ggaaca gga acg tta agc agt gga ctt agt aat tac aca gtt tca gat 624 Gly ThrGly Thr Leu Ser Ser Gly Leu Ser Asn Tyr Thr Val Ser Asp 195 200 205 accgcc tca acc act tgg ctt aca act tcc aag ctg aag gtg aag gtg 672 Thr AlaSer Thr Thr Trp Leu Thr Thr Ser Lys Leu Lys Val Lys Val 210 215 220 gataag aat cca ttc aaa ctt agt gtg tat aag cct gat gga acg acg 720 Asp LysAsn Pro Phe Lys Leu Ser Val Tyr Lys Pro Asp Gly Thr Thr 225 230 235 240ttg att gcc cgt caa tat gac agc act acg aat cgt aac att gcc tgg 768 LeuIle Ala Arg Gln Tyr Asp Ser Thr Thr Asn Arg Asn Ile Ala Trp 245 250 255tta acc aat ggc agt aca atc atc gac aag gta gaa gat cat ttt tat 816 LeuThr Asn Gly Ser Thr Ile Ile Asp Lys Val Glu Asp His Phe Tyr 260 265 270tca ccg gct tcc gag gag ttt ttt ggc ttt gga gag cat tac aac aac 864 SerPro Ala Ser Glu Glu Phe Phe Gly Phe Gly Glu His Tyr Asn Asn 275 280 285ttc cgt aaa cgc gga aat gat gtg gac acc tat gtg ttc aac cag tat 912 PheArg Lys Arg Gly Asn Asp Val Asp Thr Tyr Val Phe Asn Gln Tyr 290 295 300aag aat caa aat gac cgc acc tac atg gca att cct ttt atg ctt aac 960 LysAsn Gln Asn Asp Arg Thr Tyr Met Ala Ile Pro Phe Met Leu Asn 305 310 315320 agc agc ggt tat ggc att ttc gta aat tca acg tat tat tcc aaa ttt 1008Ser Ser Gly Tyr Gly Ile Phe Val Asn Ser Thr Tyr Tyr Ser Lys Phe 325 330335 cgg ttg gca acc gaa cgc acc gat atg ttc agc ttt acg gct gat aca 1056Arg Leu Ala Thr Glu Arg Thr Asp Met Phe Ser Phe Thr Ala Asp Thr 340 345350 ggg ggt agt gcc gcc tcg atg ctg gat tat tat ttc att tac ggt aat 1104Gly Gly Ser Ala Ala Ser Met Leu Asp Tyr Tyr Phe Ile Tyr Gly Asn 355 360365 gat ttg aaa aat gtg gtg agt aac tac gct aac att acc ggt aag cca 1152Asp Leu Lys Asn Val Val Ser Asn Tyr Ala Asn Ile Thr Gly Lys Pro 370 375380 aca gcg ctg ccg aaa tgg gct ttc ggg tta tgg atg tca gct aac gag 1200Thr Ala Leu Pro Lys Trp Ala Phe Gly Leu Trp Met Ser Ala Asn Glu 385 390395 400 tgg gat cgt caa acc aag gtg aat aca gcc att aat aac gcg aac tcc1248 Trp Asp Arg Gln Thr Lys Val Asn Thr Ala Ile Asn Asn Ala Asn Ser 405410 415 aat aat att ccg gct aca gcg gtt gtg ctc gaa cag tgg agt gat gag1296 Asn Asn Ile Pro Ala Thr Ala Val Val Leu Glu Gln Trp Ser Asp Glu 420425 430 aac acg ttt tat att ttc aat gat gcc acc tat acc ccg aaa acg ggc1344 Asn Thr Phe Tyr Ile Phe Asn Asp Ala Thr Tyr Thr Pro Lys Thr Gly 435440 445 agt gct gcg cat gcc tat acc gat ttc act ttc ccg aca tct ggg aga1392 Ser Ala Ala His Ala Tyr Thr Asp Phe Thr Phe Pro Thr Ser Gly Arg 450455 460 tgg acg gat cca aaa gcg atg gca gac aat gtg cat aac aat ggg atg1440 Trp Thr Asp Pro Lys Ala Met Ala Asp Asn Val His Asn Asn Gly Met 465470 475 480 aag ctg gtg ctt tgg cag gtc cct att cag aaa tgg act tca acgccc 1488 Lys Leu Val Leu Trp Gln Val Pro Ile Gln Lys Trp Thr Ser Thr Pro485 490 495 tat acc cag aaa gat aat gat gaa gcc tat atg acg gct cag aattat 1536 Tyr Thr Gln Lys Asp Asn Asp Glu Ala Tyr Met Thr Ala Gln Asn Tyr500 505 510 gca gtt ggc aac ggt agc gga ggc cag tac agg ata cct tca ggacaa 1584 Ala Val Gly Asn Gly Ser Gly Gly Gln Tyr Arg Ile Pro Ser Gly Gln515 520 525 tgg ttc gag aac agt ttg ctg ctt gat ttt acg aat acg gcc gccaaa 1632 Trp Phe Glu Asn Ser Leu Leu Leu Asp Phe Thr Asn Thr Ala Ala Lys530 535 540 aac tgg tgg atg tct aaa cgc gct tat ctg ttt gat ggt gtg ggtatc 1680 Asn Trp Trp Met Ser Lys Arg Ala Tyr Leu Phe Asp Gly Val Gly Ile545 550 555 560 gac ggc ttc aaa aca gat ggc ggt gaa atg gta tgg ggt cgctca aat 1728 Asp Gly Phe Lys Thr Asp Gly Gly Glu Met Val Trp Gly Arg SerAsn 565 570 575 act ttc tca aac ggt aag aaa ggc aat gaa atg cgc aat caatac ccg 1776 Thr Phe Ser Asn Gly Lys Lys Gly Asn Glu Met Arg Asn Gln TyrPro 580 585 590 aat gag tat gtg aaa gcc tat aac gag tac gcg cgc tcg aagaaa gcc 1824 Asn Glu Tyr Val Lys Ala Tyr Asn Glu Tyr Ala Arg Ser Lys LysAla 595 600 605 gat gcg gtc tcc ttt agc cgt tcc ggc acg caa ggc gca caggcg aat 1872 Asp Ala Val Ser Phe Ser Arg Ser Gly Thr Gln Gly Ala Gln AlaAsn 610 615 620 cag att ttc tgg tcc ggt gac caa gag tcg acg ttt ggt gctttt caa 1920 Gln Ile Phe Trp Ser Gly Asp Gln Glu Ser Thr Phe Gly Ala PheGln 625 630 635 640 caa gct gtg aat gca ggg ctt acg gca agt atg tct ggcgtt cct tat 1968 Gln Ala Val Asn Ala Gly Leu Thr Ala Ser Met Ser Gly ValPro Tyr 645 650 655 tgg agc tgg gat atg gca ggc ttt aca ggc act tat ccaacg gct gag 2016 Trp Ser Trp Asp Met Ala Gly Phe Thr Gly Thr Tyr Pro ThrAla Glu 660 665 670 ttg tac aaa cgt gct act gaa atg gct gct ttt gca ccggtc atg cag 2064 Leu Tyr Lys Arg Ala Thr Glu Met Ala Ala Phe Ala Pro ValMet Gln 675 680 685 ttt cat tcc gag tct aac ggc agc tct ggt atc aac gaggaa cgt tct 2112 Phe His Ser Glu Ser Asn Gly Ser Ser Gly Ile Asn Glu GluArg Ser 690 695 700 cca tgg aac gca caa gcg cgt aca ggc gac aat acg atcatt agt cat 2160 Pro Trp Asn Ala Gln Ala Arg Thr Gly Asp Asn Thr Ile IleSer His 705 710 715 720 ttt gcc aaa tat acg aat acg cgc atg aat ttg cttcct tat att tat 2208 Phe Ala Lys Tyr Thr Asn Thr Arg Met Asn Leu Leu ProTyr Ile Tyr 725 730 735 agc gaa gcg aag atg gct agt gat act ggc gtt cccatg atg cgc gcc 2256 Ser Glu Ala Lys Met Ala Ser Asp Thr Gly Val Pro MetMet Arg Ala 740 745 750 atg gcg ctt gaa tat ccg aag gac acg aac acg tacggt ttg aca caa 2304 Met Ala Leu Glu Tyr Pro Lys Asp Thr Asn Thr Tyr GlyLeu Thr Gln 755 760 765 cag tat atg ttc gga ggt aat tta ctt att gct cctgtt atg aat cag 2352 Gln Tyr Met Phe Gly Gly Asn Leu Leu Ile Ala Pro ValMet Asn Gln 770 775 780 gga gaa aca aac aag agt att tat ctt ccg cag ggggat tgg atc gat 2400 Gly Glu Thr Asn Lys Ser Ile Tyr Leu Pro Gln Gly AspTrp Ile Asp 785 790 795 800 ttc tgg ttc ggt gct cag cgt cct ggc ggt cgaaca atc agc tac acg 2448 Phe Trp Phe Gly Ala Gln Arg Pro Gly Gly Arg ThrIle Ser Tyr Thr 805 810 815 gcc ggc atc gat gat cta ccg gtt ttt gtg aagttt ggc agt att ctt 2496 Ala Gly Ile Asp Asp Leu Pro Val Phe Val Lys PheGly Ser Ile Leu 820 825 830 ccg atg aat ttg aac gcg caa tat caa gtg ggcggg acc att ggc aac 2544 Pro Met Asn Leu Asn Ala Gln Tyr Gln Val Gly GlyThr Ile Gly Asn 835 840 845 agc ttg acg agc tac acg aat ctc gcg ttc cgcatt tat ccg ctt ggg 2592 Ser Leu Thr Ser Tyr Thr Asn Leu Ala Phe Arg IleTyr Pro Leu Gly 850 855 860 aca aca acg tac gac tgg aat gat gat att ggcggt tcg gtg aaa acc 2640 Thr Thr Thr Tyr Asp Trp Asn Asp Asp Ile Gly GlySer Val Lys Thr 865 870 875 880 ata act tct aca gag caa tat ggg ttg aataaa gaa acc gtg act gtt 2688 Ile Thr Ser Thr Glu Gln Tyr Gly Leu Asn LysGlu Thr Val Thr Val 885 890 895 cca gcg att aat tct acc aag aca ttg caagtg ttt acg act aag cct 2736 Pro Ala Ile Asn Ser Thr Lys Thr Leu Gln ValPhe Thr Thr Lys Pro 900 905 910 tcc tct gta acg gtg ggt ggt tct gtg atgaca gag tac agt act tta 2784 Ser Ser Val Thr Val Gly Gly Ser Val Met ThrGlu Tyr Ser Thr Leu 915 920 925 act gcc cta acg gga gcg tcg aca ggc tggtac tat gat act gta cag 2832 Thr Ala Leu Thr Gly Ala Ser Thr Gly Trp TyrTyr Asp Thr Val Gln 930 935 940 aaa ttc act tac gtc aag ctt ggt tca agtgca tct gct caa tcc gtt 2880 Lys Phe Thr Tyr Val Lys Leu Gly Ser Ser AlaSer Ala Gln Ser Val 945 950 955 960 gtg cta aat ggc gtt aat aag gtg gaatat gaa gca gaa ttc ggc gtg 2928 Val Leu Asn Gly Val Asn Lys Val Glu TyrGlu Ala Glu Phe Gly Val 965 970 975 caa agc ggc gtt tca acg aac acg aaccat gca ggt tat act ggt aca 2976 Gln Ser Gly Val Ser Thr Asn Thr Asn HisAla Gly Tyr Thr Gly Thr 980 985 990 gga ttt gtg gac ggc ttt gag act cttgga gac aat gtt gct ttt gat 3024 Gly Phe Val Asp Gly Phe Glu Thr Leu GlyAsp Asn Val Ala Phe Asp 995 1000 1005 gtt tcc gtc aaa gcc gca ggt acttat acg atg aag gtt cgg tat 3069 Val Ser Val Lys Ala Ala Gly Thr Tyr ThrMet Lys Val Arg Tyr 1010 1015 1020 tca tcc ggt gca ggc aat ggc tca agagcc atc tat gtg aat aac 3114 Ser Ser Gly Ala Gly Asn Gly Ser Arg Ala IleTyr Val Asn Asn 1025 1030 1035 acc aaa gtg acg gac ctt gcc ttg ccg caaaca aca agc tgg gat 3159 Thr Lys Val Thr Asp Leu Ala Leu Pro Gln Thr ThrSer Trp Asp 1040 1045 1050 aca tgg ggg act gct acg ttt agc gtc tcg ctgagt aca ggt ctc 3204 Thr Trp Gly Thr Ala Thr Phe Ser Val Ser Leu Ser ThrGly Leu 1055 1060 1065 aac acg gtg aaa gtc agc tat gat ggt acc agt tcactt ggc att 3249 Asn Thr Val Lys Val Ser Tyr Asp Gly Thr Ser Ser Leu GlyIle 1070 1075 1080 aat ttc gat aac atc gcg att gta gag caa taa 3282 AsnPhe Asp Asn Ile Ala Ile Val Glu Gln 1085 1090 2 3855 DNA Bacillusglobisporus CDS (1)..(3855) sig_peptide (1)..(105) 2 atg cgt cca cca aacaaa gaa att cca cgt att ctt gct ttt ttt aca 48 Met Arg Pro Pro Asn LysGlu Ile Pro Arg Ile Leu Ala Phe Phe Thr 1 5 10 15 gcg ttt acg ttg tttggt tca acc ctt gcc ttg ctt cct gct ccg cct 96 Ala Phe Thr Leu Phe GlySer Thr Leu Ala Leu Leu Pro Ala Pro Pro 20 25 30 gcg cat gcc tat gtc agcagc cta gga aat ctc att tct tcg agt gtc 144 Ala His Ala Tyr Val Ser SerLeu Gly Asn Leu Ile Ser Ser Ser Val 35 40 45 acc gga gat acc ttg acg ctaact gtt gat aac ggt gcg gag ccg agt 192 Thr Gly Asp Thr Leu Thr Leu ThrVal Asp Asn Gly Ala Glu Pro Ser 50 55 60 gat gac ctc ttg att gtt caa gcggtg caa aac ggt att ttg aag gtg 240 Asp Asp Leu Leu Ile Val Gln Ala ValGln Asn Gly Ile Leu Lys Val 65 70 75 80 gat tat cgt cca aat agc ata acgccg agc gcg aag acg ccg atg ctg 288 Asp Tyr Arg Pro Asn Ser Ile Thr ProSer Ala Lys Thr Pro Met Leu 85 90 95 gat ccg aac aaa act tgg tca gct gtagga gct acg att aat acg aca 336 Asp Pro Asn Lys Thr Trp Ser Ala Val GlyAla Thr Ile Asn Thr Thr 100 105 110 gcc aat cca atg acc atc acg act tccaat atg aag att gag att acc 384 Ala Asn Pro Met Thr Ile Thr Thr Ser AsnMet Lys Ile Glu Ile Thr 115 120 125 aag aat cca gta cga atg acg gtc aagaag gcg gac ggc act acg cta 432 Lys Asn Pro Val Arg Met Thr Val Lys LysAla Asp Gly Thr Thr Leu 130 135 140 ttc tgg gaa cca tca ggc gga ggg gtattc tca gac ggt gtg cgc ttc 480 Phe Trp Glu Pro Ser Gly Gly Gly Val PheSer Asp Gly Val Arg Phe 145 150 155 160 ctt cat gcc aca ggg gat aat atgtat ggc atc cgg agc ttc aat gct 528 Leu His Ala Thr Gly Asp Asn Met TyrGly Ile Arg Ser Phe Asn Ala 165 170 175 ttt gat agc ggg ggt gac ctg ctgcgg aat tcg tcc aat cat gcc gcc 576 Phe Asp Ser Gly Gly Asp Leu Leu ArgAsn Ser Ser Asn His Ala Ala 180 185 190 cat gcg ggt gaa cag gga gat tccggt ggt ccg ctt att tgg agt acg 624 His Ala Gly Glu Gln Gly Asp Ser GlyGly Pro Leu Ile Trp Ser Thr 195 200 205 gca gga tat gga cta tta gtc gatagc gat ggc ggc tac ccc tat aca 672 Ala Gly Tyr Gly Leu Leu Val Asp SerAsp Gly Gly Tyr Pro Tyr Thr 210 215 220 gat agc aca acc ggt caa atg gagttt tat tat ggt ggg acc cct cct 720 Asp Ser Thr Thr Gly Gln Met Glu PheTyr Tyr Gly Gly Thr Pro Pro 225 230 235 240 gag gga cgt cgt tat gcg aaacaa aac gtg gaa tat tat att atg ctc 768 Glu Gly Arg Arg Tyr Ala Lys GlnAsn Val Glu Tyr Tyr Ile Met Leu 245 250 255 gga acc ccc aag gaa att atgacc gac gta ggg gaa atc aca ggg aaa 816 Gly Thr Pro Lys Glu Ile Met ThrAsp Val Gly Glu Ile Thr Gly Lys 260 265 270 ccg cct atg ctg cct aag tggtcg ctt gga ttc atg aac ttt gag tgg 864 Pro Pro Met Leu Pro Lys Trp SerLeu Gly Phe Met Asn Phe Glu Trp 275 280 285 gat acg aat caa acg gag tttacg aat aat gtg gat acg tat cgt gcc 912 Asp Thr Asn Gln Thr Glu Phe ThrAsn Asn Val Asp Thr Tyr Arg Ala 290 295 300 aaa aat atc ccc ata gat gcttac gcc ttc gac tat gac tgg aaa aag 960 Lys Asn Ile Pro Ile Asp Ala TyrAla Phe Asp Tyr Asp Trp Lys Lys 305 310 315 320 tac ggg gaa acc aac tatggt gaa ttc gcg tgg aat acg act aat ttc 1008 Tyr Gly Glu Thr Asn Tyr GlyGlu Phe Ala Trp Asn Thr Thr Asn Phe 325 330 335 cct tct gcg tca acg acttct tta aag tca aca atg gat gct aaa ggc 1056 Pro Ser Ala Ser Thr Thr SerLeu Lys Ser Thr Met Asp Ala Lys Gly 340 345 350 atc aaa atg atc gga attaca aaa ccc cgc atc gtt acg aag gat gct 1104 Ile Lys Met Ile Gly Ile ThrLys Pro Arg Ile Val Thr Lys Asp Ala 355 360 365 tca gcg aat gtg acg acccaa ggg acg gac gcg aca aat ggc ggt tat 1152 Ser Ala Asn Val Thr Thr GlnGly Thr Asp Ala Thr Asn Gly Gly Tyr 370 375 380 ttt tat cca ggc cat aacgag tat cag gat tat ttc att ccc gta act 1200 Phe Tyr Pro Gly His Asn GluTyr Gln Asp Tyr Phe Ile Pro Val Thr 385 390 395 400 gtg cgt agt atc gatcct tac aat gct aac gaa cgt gct tgg ttc tgg 1248 Val Arg Ser Ile Asp ProTyr Asn Ala Asn Glu Arg Ala Trp Phe Trp 405 410 415 aat cat tcc aca gatgcg ctt aat aaa ggg atc gta ggt tgg tgg aat 1296 Asn His Ser Thr Asp AlaLeu Asn Lys Gly Ile Val Gly Trp Trp Asn 420 425 430 gac gag acg gat aaagta tct tcg ggt gga gcg tta tat tgg ttt ggc 1344 Asp Glu Thr Asp Lys ValSer Ser Gly Gly Ala Leu Tyr Trp Phe Gly 435 440 445 aat ttc aca aca ggccac atg tct cag acg atg tac gaa ggg ggg cgg 1392 Asn Phe Thr Thr Gly HisMet Ser Gln Thr Met Tyr Glu Gly Gly Arg 450 455 460 gct tac acg agt ggagcg cag cgt gtt tgg caa acg gct aga acc ttc 1440 Ala Tyr Thr Ser Gly AlaGln Arg Val Trp Gln Thr Ala Arg Thr Phe 465 470 475 480 tac cca ggt gcccag cgg tat gcg act acg ctt tgg tct ggc gat att 1488 Tyr Pro Gly Ala GlnArg Tyr Ala Thr Thr Leu Trp Ser Gly Asp Ile 485 490 495 ggc att caa tacaat aaa ggc gaa cgg atc aat tgg gct gcc ggg atg 1536 Gly Ile Gln Tyr AsnLys Gly Glu Arg Ile Asn Trp Ala Ala Gly Met 500 505 510 cag gag caa agggca gtt atg cta tcc tcc gtg aac aat ggc cag gtg 1584 Gln Glu Gln Arg AlaVal Met Leu Ser Ser Val Asn Asn Gly Gln Val 515 520 525 aaa tgg ggc atggat acc ggc gga ttc aat cag cag gat ggc acg acg 1632 Lys Trp Gly Met AspThr Gly Gly Phe Asn Gln Gln Asp Gly Thr Thr 530 535 540 aac aat ccg aatccc gat tta tac gct cgg tgg atg cag ttc agt gcc 1680 Asn Asn Pro Asn ProAsp Leu Tyr Ala Arg Trp Met Gln Phe Ser Ala 545 550 555 560 cta acg cctgtt ttc cga gtg cat ggg aac aac cat cag cag cgc cag 1728 Leu Thr Pro ValPhe Arg Val His Gly Asn Asn His Gln Gln Arg Gln 565 570 575 cca tgg tacttc gga tcg act gcg gag gag gcc tcc aaa gag gca att 1776 Pro Trp Tyr PheGly Ser Thr Ala Glu Glu Ala Ser Lys Glu Ala Ile 580 585 590 cag ctg cggtac tcc ctg atc cct tat atg tat gcc tat gag aga agt 1824 Gln Leu Arg TyrSer Leu Ile Pro Tyr Met Tyr Ala Tyr Glu Arg Ser 595 600 605 gct tac gagaat ggg aat ggg ctc gtt cgg cca ttg atg caa gcc tat 1872 Ala Tyr Glu AsnGly Asn Gly Leu Val Arg Pro Leu Met Gln Ala Tyr 610 615 620 cca aca gatgcg gcc gtc aaa aat tac acg gat gct tgg atg ttt ggt 1920 Pro Thr Asp AlaAla Val Lys Asn Tyr Thr Asp Ala Trp Met Phe Gly 625 630 635 640 gac tggctg ctg gct gca cct gtg gta gat aaa cag cag acg agt aag 1968 Asp Trp LeuLeu Ala Ala Pro Val Val Asp Lys Gln Gln Thr Ser Lys 645 650 655 gat atctat tta ccg tct ggg tca tgg att gac tat gcg cga ggc aat 2016 Asp Ile TyrLeu Pro Ser Gly Ser Trp Ile Asp Tyr Ala Arg Gly Asn 660 665 670 gca ataact ggc ggt caa acc atc cga tat tcg gtt aat ccg gac acg 2064 Ala Ile ThrGly Gly Gln Thr Ile Arg Tyr Ser Val Asn Pro Asp Thr 675 680 685 ttg acagac atg cct ctc ttt att aaa aaa ggt gcc att att cca aca 2112 Leu Thr AspMet Pro Leu Phe Ile Lys Lys Gly Ala Ile Ile Pro Thr 690 695 700 cag aaagtg cag gat tac gta ggg cag gct tcc gtc act tcc gtt gat 2160 Gln Lys ValGln Asp Tyr Val Gly Gln Ala Ser Val Thr Ser Val Asp 705 710 715 720 gtggat gtg ttt ccg gat acg acg cag tcg agt ttc acg tac tac gat 2208 Val AspVal Phe Pro Asp Thr Thr Gln Ser Ser Phe Thr Tyr Tyr Asp 725 730 735 gatgat ggc gcc agt tat aac tat gag agc ggc act tat ttt aag caa 2256 Asp AspGly Ala Ser Tyr Asn Tyr Glu Ser Gly Thr Tyr Phe Lys Gln 740 745 750 aatatg act gct cag gat aat ggg tca ggc tcg tta agt ttt act tta 2304 Asn MetThr Ala Gln Asp Asn Gly Ser Gly Ser Leu Ser Phe Thr Leu 755 760 765 ggagca aag agt ggc agt tac acg ccg gct ctc caa tcc tat atc gtt 2352 Gly AlaLys Ser Gly Ser Tyr Thr Pro Ala Leu Gln Ser Tyr Ile Val 770 775 780 aagctg cac ggt tct gct gga act tct gtt acg aat aac agc gca gct 2400 Lys LeuHis Gly Ser Ala Gly Thr Ser Val Thr Asn Asn Ser Ala Ala 785 790 795 800atg aca tct tat gca agc ttg gaa gca tta aaa gct gct gct ggg gaa 2448 MetThr Ser Tyr Ala Ser Leu Glu Ala Leu Lys Ala Ala Ala Gly Glu 805 810 815ggc tgg gcg act ggg aag gac att tat ggg gat gtc acc tat gtg aaa 2496 GlyTrp Ala Thr Gly Lys Asp Ile Tyr Gly Asp Val Thr Tyr Val Lys 820 825 830gtg acg gca ggt aca gct tct tct aaa tct att gct gtt aca ggt gtt 2544 ValThr Ala Gly Thr Ala Ser Ser Lys Ser Ile Ala Val Thr Gly Val 835 840 845gct gcc gtg agc gca act act tcg caa tac gaa gct gag gat gca tcg 2592 AlaAla Val Ser Ala Thr Thr Ser Gln Tyr Glu Ala Glu Asp Ala Ser 850 855 860ctt tct ggc aat tcg gtt gct gca aag gcg tcc ata aac acg aat cat 2640 LeuSer Gly Asn Ser Val Ala Ala Lys Ala Ser Ile Asn Thr Asn His 865 870 875880 acc gga tat acg gga act gga ttt gta gat ggt ttg ggg aat gat ggc 2688Thr Gly Tyr Thr Gly Thr Gly Phe Val Asp Gly Leu Gly Asn Asp Gly 885 890895 gct ggt gtc acc ttc tat cca aag gtg aaa act ggc ggt gac tac aat 2736Ala Gly Val Thr Phe Tyr Pro Lys Val Lys Thr Gly Gly Asp Tyr Asn 900 905910 gtc tcc ttg cgt tat gcg aat gct tca ggc acg gct aag tca gtc agt 2784Val Ser Leu Arg Tyr Ala Asn Ala Ser Gly Thr Ala Lys Ser Val Ser 915 920925 att ttt gtt aat gga aaa aga gtg aag tcc acc tcg ctc gct aat ctc 2832Ile Phe Val Asn Gly Lys Arg Val Lys Ser Thr Ser Leu Ala Asn Leu 930 935940 gca aat tgg gac act tgg tct aca caa tct gag aca ctg ccg ttg acg 2880Ala Asn Trp Asp Thr Trp Ser Thr Gln Ser Glu Thr Leu Pro Leu Thr 945 950955 960 gca ggt gtg aat gtt gtg acc tat aaa tat tac tcc gat gcg gga gat2928 Ala Gly Val Asn Val Val Thr Tyr Lys Tyr Tyr Ser Asp Ala Gly Asp 965970 975 aca ggc aat gtt aac atc gac aac atc acg gta cct ttt gcg cca att2976 Thr Gly Asn Val Asn Ile Asp Asn Ile Thr Val Pro Phe Ala Pro Ile 980985 990 atc ggt aag tat gaa gca gag agt gct gag ctt tct ggt ggc agc tca3024 Ile Gly Lys Tyr Glu Ala Glu Ser Ala Glu Leu Ser Gly Gly Ser Ser 9951000 1005 ttg aac acg aac cat tgg tac tac agt ggt acg gct ttt gta gac3069 Leu Asn Thr Asn His Trp Tyr Tyr Ser Gly Thr Ala Phe Val Asp 10101015 1020 ggt ttg agt gct gta ggc gcg cag gtg aaa tac aac gtg aat gtc3114 Gly Leu Ser Ala Val Gly Ala Gln Val Lys Tyr Asn Val Asn Val 10251030 1035 cct agc gca gga agt tat cag gta gcg ctg cga tat gcg aat ggc3159 Pro Ser Ala Gly Ser Tyr Gln Val Ala Leu Arg Tyr Ala Asn Gly 10401045 1050 agt gca gcg acg aaa acg ttg agt act tat atc aat gga gcc aag3204 Ser Ala Ala Thr Lys Thr Leu Ser Thr Tyr Ile Asn Gly Ala Lys 10551060 1065 ctg ggg caa acc agt ttt acg agt cct ggt acg aat tgg aat gtt3249 Leu Gly Gln Thr Ser Phe Thr Ser Pro Gly Thr Asn Trp Asn Val 10701075 1080 tgg cag gat aat gtg caa acg gtg acg tta aat gca ggg gca aac3294 Trp Gln Asp Asn Val Gln Thr Val Thr Leu Asn Ala Gly Ala Asn 10851090 1095 acg att gcg ttt aaa tac gac gcc gct gac agc ggg aac atc aac3339 Thr Ile Ala Phe Lys Tyr Asp Ala Ala Asp Ser Gly Asn Ile Asn 11001105 1110 gta gat cgt ctg ctt ctt tca act tcg gca gcg gga acg ccg gtt3384 Val Asp Arg Leu Leu Leu Ser Thr Ser Ala Ala Gly Thr Pro Val 11151120 1125 tct gag cag aac ctg cta gac aat ccc ggt ttc gag cgt gac acg3429 Ser Glu Gln Asn Leu Leu Asp Asn Pro Gly Phe Glu Arg Asp Thr 11301135 1140 agt caa acc aat aac tgg att gag tgg cat cca ggc acg caa gct3474 Ser Gln Thr Asn Asn Trp Ile Glu Trp His Pro Gly Thr Gln Ala 11451150 1155 gtt gct ttt ggc gtt gat agc ggc tca acc acc aat ccg ccg gaa3519 Val Ala Phe Gly Val Asp Ser Gly Ser Thr Thr Asn Pro Pro Glu 11601165 1170 tcc ccg tgg tcg ggt gat aag cgt gcc tac ttc ttt gca gca ggt3564 Ser Pro Trp Ser Gly Asp Lys Arg Ala Tyr Phe Phe Ala Ala Gly 11751180 1185 gcc tat caa caa agc atc cat caa acc att agt gtt cct gtt aat3609 Ala Tyr Gln Gln Ser Ile His Gln Thr Ile Ser Val Pro Val Asn 11901195 1200 aat gta aaa tac aaa ttt gaa gcc tgg gtc cgc atg aag aat acg3654 Asn Val Lys Tyr Lys Phe Glu Ala Trp Val Arg Met Lys Asn Thr 12051210 1215 acg ccg acg acg gca aga gcc gaa att caa aac tat ggc gga tca3699 Thr Pro Thr Thr Ala Arg Ala Glu Ile Gln Asn Tyr Gly Gly Ser 12201225 1230 gcc att tat gcg aac ata agt aac agc ggt gtt tgg aaa tat atc3744 Ala Ile Tyr Ala Asn Ile Ser Asn Ser Gly Val Trp Lys Tyr Ile 12351240 1245 agc gta agt gat att atg gtg acc aat ggt cag ata gat gtt gga3789 Ser Val Ser Asp Ile Met Val Thr Asn Gly Gln Ile Asp Val Gly 12501255 1260 ttt tac gtg gat tca cct ggt gga act acg ctt cac att gat gat3834 Phe Tyr Val Asp Ser Pro Gly Gly Thr Thr Leu His Ile Asp Asp 12651270 1275 gtg cgc gta acc aaa caa taa 3855 Val Arg Val Thr Lys Gln 1280

1. A branched cyclotetrasaccharide which is a glycosyl derivative ofcyclotetrasaccharide represented bycyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→},and which has a structure represented by Formula 1; Formula 1:

wherein in Formula 1, R₁ to R₁₂ each independently represents anoptionally substituted glycosyl group or hydrogen atom, with the provisothat when all of R₁ to R₁₂ are not hydrogen atom and either R₄ or R₁₀ isan optionally substituted glycosyl group, R₄ or R₁₀ as the glycosylgroup is a member selected from the group consisting of glycosyl groupsother than D-glucopyranosyl group.
 2. The branched cyclotetrasaccharideof claim 1, wherein one or more glycosyl groups positioning at one ormore positions of R₁ to R₁₂ in Formula 1 each independently representany one of the glycosyl groups selected from those represented by thefollowing groups of (1) to (5): (1) optionally substituted{α-D-glucopyranosyl-(1→4)-}_(n) α-D-glucopyranosyl groups, with theproviso that each “n” in the above groups independently means an integerof 0 or more when at least two of R₁ to R₁₂ are the above groups; (2)optionally substitutedα-D-glucopyranosyl-(1→6)-{α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-})_(n)α-D-glucopyranosyl groups, with the proviso that each “n” in the abovegroups independently means an integer of 0 or more when at least two ofR₁ to R₁₂ are the above groups; (3) optionally substituted{β-D-galactopyranosyl-(1→6)-})_(n)β-D-galactopyranosyl groups, with theproviso that each “n” in the above groups independently means an integerof 0 or when at least two of R₁ to R₁₂ are the above groups; (4)optionally substituted α-D-galactopyranosyl groups; and (5) optionallysubstituted β-D-chitosaminyl groups.
 3. The branchedcyclotetrasaccharide of claim 1 or 2, wherein R₁ and/or R₇ in Formula 1are independently optionally substituted {α-D-glucopyranosyl-(1→4)-}_(n)α-D-glucopyranosyl groups, with the proviso that each “n” in the abovegroups independently means an integer of 0 or more when both R₁ and R₇are the above groups.
 4. The branched cyclotetrasaccharide of claim 3,which is the one represented by Chemical Formula 1 or 2;


5. The branched cyclotetrasaccharide of claim 1, 2 or 3, wherein R₂and/or R₈ in Formula 1 are optionally substitutedα-D-glucopyranosyl-(1→6)-{α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-}_(n)α-D-glucopyranosyl groups, with the proviso that each “n” in the abovegroups independently means an integer of 0 or more when both R₂ and R₈are the above groups.
 6. The branched cyclotetrasaccharide of claim 5,which is the one represented by Chemical Formula 3, 4, or 5;


7. The branched cyclotetrasaccharide of any one of claims 1 to 3,wherein R₂ and/or R₈ in Formula 1 are optionally substituted{β-D-galactopyranosyl-(1→6)-}_(n)β-D-galactopyranosyl groups, with theproviso that each “n” in the above groups independently means an integerof 0 or more when both R₂ and R₈ are the above groups.
 8. The branchedcyclotetrasaccharide of claim 7, which is the one represented byChemical Formula 6;


9. The branched cyclotetrasaccharide of claim 1, 2 or 3, wherein R₄and/or R₁₀ in Formula 1 are optionally substituted{β-D-galactopyranosyl-(1→6)-}_(n) β-D-galactopyranosyl groups, with theproviso that each “n” in the above groups independently means an integerof 0 or more when both R₄ and R₁₀ are the groups.
 10. The branchedcyclotetrasaccharide of claim 7, which is the one represented byChemical Formula 7 or 8:


11. The branched cyclotetrasaccharide of claim 1, 2 or 3, wherein R₄and/or R₁₀ in Formula 1 are optionally substituted α-D-galactopyranosylgroups.
 12. The branched cyclotetrasaccharide of claim 11, which is theone represented by Chemical Formula 9;


13. The branched cyclotetrasaccharide of claim 1, 2 or 3, wherein R,and/or R, in Formula 1 are optionally substituted β-D-chitosaminylgroups.
 14. The branched cyclotetrasaccharide of claim 13, which is theone represented by Chemical Formula 10; Chemical Formula 10:


15. The branched cyclotetrasaccharide of any one of claims 1 to 14,which is in the form a solution, amorphous powder, or molasses.
 16. Anisolated crystal of the branched cyclotetrasaccharide of any one ofclaims 1 to
 14. 17. The isolated crystal of the branchedcyclotetrasaccharide of claim 16, which is a crystal of acyclotetrasaccharide, represented by Chemical Formula 1, 2, 3, 6, or 7.18. The isolated crystal of the branched cyclotetrasaccharide of claim16 or 17, which is in the form of a hydrous- or anhydrous-crystal. 19.The isolated crystal of the branched cyclotetrasaccharide of claim 16,17 or 18, which has main diffraction angles (2θ) of any one of (1) to(5) on X-ray powder diffraction analysis; (1) 8.1°, 12.2°, 14.2°, and15.4°; (2) 5.6°, 8.8°, 16.9°, and 21.9°; (3) 7.9°, 12.1°, 17.9°, and20.2°; (4) 11.0°, 12.3°, 12.8°, and 24.9°; and (5) 8.7°, 13.0°, 21.7°,and 26.1°.


20. A saccharide composition comprising the branchedcyclotetrasaccharide of any one of claims 1 to 14 and othersaccharide(s) except for the branched cyclotetrasaccharide.
 21. Thesaccharide composition of claim 20, which is in the form of a solution,amorphous powder, molasses, or crystalline powder.
 22. A process forproducing a branched cyclotetrasaccharide which uses the action of anenzyme capable of transferring a glycosyl group from a monosaccharide,oligosaccharide, or polysaccharide to cyclotetrasaccharide representedbycyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→},and which is characterized in that it comprises the following two stepsof; (1) forming the branched cyclotetrasaccharide of anyone of claims 1to 14 by allowing the enzyme to act on a mixture of thecyclotetrasaccharide along with the monosaccharide, the oligosaccharide,or the polysaccharide, and (2) collecting the formed branchedcyclotetrasaccharide in the step (1).
 23. The process of claim 22, whichfurther contains the following step for producing thecyclotetrasaccharide prior to the step (1); forming thecyclotetrasaccharide by allowing an α-isomaltosylglucosaccharide-formingenzyme, having the following enzymatic activity (A) and anα-isomaltosyl-transferring enzyme having the following enzymaticactivity (B), to act on a saccharide having both a glucosepolymerization degree of at least two and the α-1,4 glucosyl bond as alinkage at the non-reducing end, and collecting the formedcyclotetrasaccharide; Enzymatic activity (A): Acting on a saccharide,having both a glucose polymerization degree of “n” (“n” is an integer oftwo or more) and the α-1,4 glucosyl bond as a linkage at thenon-reducing end, and forming a saccharide having a glucosepolymerization degree of “n+1” and having both the α-1,6 glucosyl bondas a linkage at the non-reducing end and the α-1,4 glucosyl bond as alinkage other than the non-reducing end, without substantiallyincreasing the reducing power; and Enzymatic activity (B): Acting on asaccharide, having a glucose polymerization degree of at least three andhaving both the α-1,6 glucosyl bond as a linkage at the non-reducing endand the α-1,4 glucosyl bond as a linkage other than the non-reducingend, and forming cyclotetrasaccharide represented bycyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}.24. The process for producing a branched cyclotetrasaccharide of claim22 or 23, which uses one or more enzymes selected from the groupconsisting of cyclomaltodextrin glucanotransferase, β-galactosidase,α-galactosidase, lysozyme, an α-isomaltosylglucosaccharide-formingenzyme having the following enzymatic activity (A), and anα-isomaltosyl-transferring enzyme having the following enzymaticactivity (B), which are enzymes capable of transferring a glycosyl groupfrom a monosaccharide, oligosaccharide, or polysaccharide tocyclotetrasaccharide; Enzymatic activity (A): Acting on a saccharide,having the 1,4-glucosyl bond and a glucose polymerization degree of “n”(“n” is an integer of two or more, and forming a saccharide, having aglucose polymerization degree of “n+1” and having both the α-1,6glucosyl bond as a linkage at the non-reducing end and the α-1,4glucosyl bond as a linkage other than the non-reducing end, withoutsubstantially increasing the reducing power, and Enzymatic activity (B):Acting on a saccharide, having a glucose polymerization degree of atleast three and having both the α-1,6 glucosyl bond as a linkage at thenon-reducing end and the α-1,4 glucosyl bond as a linkage other than thenon-reducing end, and forming a cyclotetrasaccharide represented bycyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→)}.25. The process of claim 22, 23, or 24, which uses, as themonosaccharide, oligosaccharide or polysaccharide, one or moresaccharides selected from the group consisting of glucose-1-phospate,maltooligosaccharide, circular dextrin, panose,isomaltosylglucosaccharide, lactose, melibiose, N-acetylchitooligosaccharide, dextrin, glycogen, liquefied starch, and chitin asa monosaccharide, oligosaccharide, and polysaccharide.
 26. The processof any one of claims 22 to 25, wherein the formed branchedcyclotetrasaccharide is collected by a step comprising one or morepurification methods selected from the group consisting of decoloration,desalting, column chromatography, and crystallization.
 27. A method fortransferring a glycosyl group using an enzyme capable of transferring aglycosyl group from a monosaccharide, oligosaccharide, or polysaccharideto a cyclotetrasaccharide represented bycyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→};said method comprising a step of allowing the enzyme to act on a mixtureof cyclotetrasaccharide and the monosaccharide, oligosaccharide, orpolysaccharide to form the branched cyclotetrasaccharide of any one ofclaims 1 to
 14. 28. A composition comprising the branchedcyclotetrasaccharide of any one of claims 1 to
 14. 29. The compositionof claim 28, which is in the form of a food, cosmetic, orpharmaceutical.